Nucleic acid and corresponding protein entitled 158P3D2 useful in treatment and detection of cancer

ABSTRACT

A novel gene (designated 158P3D2) and its encoded protein, and variants thereof, are described wherein 158P3D2 exhibits tissue specific expression in normal adult tissue, and is aberrantly expressed in the cancers listed in Table I. Consequently, 158P3D2 provides a diagnostic, prognostic, prophylactic and/or therapeutic target for cancer. The 158P3D2 gene or fragment thereof, or its encoded protein, or variants thereof, or a fragment thereof, can be used to elicit a humoral or cellular immune response; antibodies or T cells reactive with 158P3D2 can be used in active or passive immunization.

CROSS-REFERENCE TO RELATED APPLICATIONS

This application is a continuation of U.S. patent application Ser. No. 10/107,532, filed Mar. 25, 2002, which claims the benefit of priority from U.S. Ser. No. 60/283,112 filed Apr. 10, 2001, now abandoned, and U.S. Ser. No. 60/286,630, filed Apr. 25, 2001, now abandoned. The contents of these applications are hereby incorporated by reference herein in their entirety.

SUBMISSION ON COMPACT DISC

The content of the following submission on compact discs is incorporated herein by reference in its entirety: A computer readable form copy of the Sequence Listing (CRF) on compact disc (file name: 511582006401, date recorded: Jul. 6, 2006, size: 1,081,344 bytes); a compact disc copy of the Sequence Listing (COPY 1) (file name: 511582006401, date recorded: Jul. 6, 2006, size: 1,081,344 bytes); and a duplicate compact disc copy of the Sequence Listing (COPY 2) (file name: 511582006401, date recorded: Jul. 6, 2006, size: 1,081,344 bytes).

FIELD OF THE INVENTION

The invention described herein relates to a gene and its encoded protein, termed 158P3D2 expressed in certain cancers, and to diagnostic and therapeutic methods and compositions useful in the management of cancers that express 158P3D2.

BACKGROUND OF THE INVENTION

Cancer is the second leading cause of human death next to coronary disease. Worldwide, millions of people die from cancer every year. In the United States alone, as reported by the American Cancer Society, cancer causes the death of well over a half-million people annually, with over 1.2 million new cases diagnosed per year. While deaths from heart disease have been declining significantly, those resulting from cancer generally are on the rise. In the early part of the next century, cancer is predicted to become the leading cause of death.

Worldwide, several cancers stand out as the leading killers. In particular, carcinomas of the lung, prostate, breast, colon, pancreas, and ovary represent the primary causes of cancer death. These and virtually all other carcinomas share a common lethal feature. With very few exceptions, metastatic disease from a carcinoma is fatal. Moreover, even for those cancer patients who initially survive their primary cancers, common experience has shown that their lives are dramatically altered. Many cancer patients experience strong anxieties driven by the awareness of the potential for recurrence or treatment failure. Many cancer patients experience physical debilitations following treatment. Furthermore, many cancer patients experience a recurrence.

Worldwide, prostate cancer is the fourth most prevalent cancer in men. In North America and Northern Europe, it is by far the most common cancer in males and is the second leading cause of cancer death in men. In the United States alone, well over 30,000 men die annually of this disease—second only to lung cancer. Despite the magnitude of these figures, there is still no effective treatment for metastatic prostate cancer. Surgical prostatectomy, radiation therapy, hormone ablation therapy, surgical castration and chemotherapy continue to be the main treatment modalities. Unfortunately, these treatments are ineffective for many and are often associated with undesirable consequences.

On the diagnostic front, the lack of a prostate tumor marker that can accurately detect early-stage, localized tumors remains a significant limitation in the diagnosis and management of this disease. Although the serum prostate specific antigen (PSA) assay has been a very useful tool, however its specificity and general utility is widely regarded as lacking in several important respects.

Progress in identifying additional specific markers for prostate cancer has been improved by the generation of prostate cancer xenografts that can recapitulate different stages of the disease in mice. The LAPC (Los Angeles Prostate Cancer) xenografts are prostate cancer xenografts that have survived passage in severe combined immune deficient (SCID) mice and have exhibited the capacity to mimic the transition from androgen dependence to androgen independence (Klein et al., 1997, Nat. Med. 3:402). More recently identified prostate cancer markers include PCTA-1 (Su et al., 1996, Proc. Natl. Acad. Sci. USA 93: 7252), prostate-specific membrane (PSM) antigen (Pinto et al., Clin. Cancer Res. 1996 Sep. 2 (9): 1445-51), STEAP (Hubert, et al., Proc Natl Acad Sci USA. 1999 Dec. 7; 96(25): 14523-8) and prostate stem cell antigen (PSCA) (Reiter et al., 1998, Proc. Natl. Acad. Sci. USA 95: 1735).

While previously identified markers such as PSA, PSM, PCTA and PSCA have facilitated efforts to diagnose and treat prostate cancer, there is need for the identification of additional markers and therapeutic targets for prostate and related cancers in order to further improve diagnosis and therapy.

Renal cell carcinoma (RCC) accounts for approximately 3 percent of adult malignancies. Once adenomas reach a diameter of 2 to 3 cm, malignant potential exists. In the adult, the two principal malignant renal tumors are renal cell adenocarcinoma and transitional cell carcinoma of the renal pelvis or ureter. The incidence of renal cell adenocarcinoma is estimated at more than 29,000 cases in the United States, and more than 11,600 patients died of this disease in 1998. Transitional cell carcinoma is less frequent, with an incidence of approximately 500 cases per year in the United States.

Surgery has been the primary therapy for renal cell adenocarcinoma for many decades. Until recently, metastatic disease has been refractory to any systemic therapy. With recent developments in systemic therapies, particularly immunotherapies, metastatic renal cell carcinoma may be approached aggressively in appropriate patients with a possibility of durable responses. Nevertheless, there is a remaining need for effective therapies for these patients.

Of all new cases of cancer in the United States, bladder cancer represents approximately 5 percent in men (fifth most common neoplasm) and 3 percent in women (eighth most common neoplasm). The incidence is increasing slowly, concurrent with an increasing older population. In 1998, there was an estimated 54,500 cases, including 39,500 in men and 15,000 in women. The age-adjusted incidence in the United States is 32 per 100,000 for men and 8 per 100,000 in women. The historic male/female ratio of 3:1 may be decreasing related to smoking patterns in women. There were an estimated 11,000 deaths from bladder cancer in 1998 (7,800 in men and 3,900 in women). Bladder cancer incidence and mortality strongly increase with age and will be an increasing problem as the population becomes more elderly.

Most bladder cancers recur in the bladder. Bladder cancer is managed with a combination of transurethral resection of the bladder (TUR) and intravesical chemotherapy or immunotherapy. The multifocal and recurrent nature of bladder cancer points out the limitations of TUR. Most muscle-invasive cancers are not cured by TUR alone. Radical cystectomy and urinary diversion is the most effective means to eliminate the cancer but carry an undeniable impact on urinary and sexual function. There continues to be a significant need for treatment modalities that are beneficial for bladder cancer patients.

An estimated 130,200 cases of colorectal cancer occurred in 2000 in the United States, including 93,800 cases of colon cancer and 36,400 of rectal cancer. Colorectal cancers are the third most common cancers in men and women. Incidence rates declined significantly during 1992-1996 (−2.1% per year). Research suggests that these declines have been due to increased screening and polyp removal, preventing progression of polyps to invasive cancers. There were an estimated 56,300 deaths (47,700 from colon cancer, 8,600 from rectal cancer) in 2000, accounting for about 11% of all U.S. cancer deaths.

At present, surgery is the most common form of therapy for colorectal cancer, and for cancers that have not spread, it is frequently curative. Chemotherapy, or chemotherapy plus radiation, is given before or after surgery to most patients whose cancer has deeply perforated the bowel wall or has spread to the lymph nodes. A permanent colostomy (creation of an abdominal opening for elimination of body wastes) is occasionally needed for colon cancer and is infrequently required for rectal cancer. There continues to be a need for effective diagnostic and treatment modalities for colorectal cancer.

There were an estimated 164,100 new cases of lung and bronchial cancer in 2000, accounting for 14% of all U.S. cancer diagnoses. The incidence rate of lung and bronchial cancer is declining significantly in men, from a high of 86.5 per 100,000 in 1984 to 70.0 in 1996. In the 1990s, the rate of increase among women began to slow. In 1996, the incidence rate in women was 42.3 per 100,000.

Lung and bronchial cancer caused an estimated 156,900 deaths in 2000, accounting for 28% of all cancer deaths. During 1992-1996, mortality from lung cancer declined significantly among men (−1.7% per year) while rates for women were still significantly increasing (0.9% per year). Since 1987, more women have died each year of lung cancer than breast cancer, which, for over 40 years, was the major cause of cancer death in women. Decreasing lung cancer incidence and mortality rates most likely resulted from decreased smoking rates over the previous 30 years; however, decreasing smoking patterns among women lag behind those of men. Of concern, although the declines in adult tobacco use have slowed, tobacco use in youth is increasing again.

Treatment options for lung and bronchial cancer are determined by the type and stage of the cancer and include surgery, radiation therapy, and chemotherapy. For many localized cancers, surgery is usually the treatment of choice. Because the disease has usually spread by the time it is discovered, radiation therapy and chemotherapy are often needed in combination with surgery. Chemotherapy alone or combined with radiation is the treatment of choice for small cell lung cancer; on this regimen, a large percentage of patients experience remission, which in some cases is long lasting. There is however, an ongoing need for effective treatment and diagnostic approaches for lung and bronchial cancers.

An estimated 182,800 new invasive cases of breast cancer were expected to occur among women in the United States during 2000. Additionally, about 1,400 new cases of breast cancer were expected to be diagnosed in men in 2000. After increasing about 4% per year in the 1980s, breast cancer incidence rates in women have leveled off in the 1990s to about 110.6 cases per 100,000.

In the U.S. alone, there were an estimated 41,200 deaths (40,800 women, 400 men) in 2000 due to breast cancer. Breast cancer ranks second among cancer deaths in women. According to the most recent data, mortality rates declined significantly during 1992-1996 with the largest decreases in younger women, both white and black. These decreases were probably the result of earlier detection and improved treatment.

Taking into account the medical circumstances and the patient's preferences, treatment of breast cancer may involve lumpectomy (local removal of the tumor) and removal of the lymph nodes under the arm; mastectomy (surgical removal of the breast) and removal of the lymph nodes under the arm; radiation therapy; chemotherapy; or hormone therapy. Often, two or more methods are used in combination. Numerous studies have shown that, for early stage disease, long-term survival rates after lumpectomy plus radiotherapy are similar to survival rates after modified radical mastectomy. Significant advances in reconstruction techniques provide several options for breast reconstruction after mastectomy. Recently, such reconstruction has been done at the same time as the mastectomy.

Local excision of ductal carcinoma in situ (DCIS) with adequate amounts of surrounding normal breast tissue may prevent the local recurrence of the DCIS. Radiation to the breast and/or tamoxifen may reduce the chance of DCIS occurring in the remaining breast tissue. This is important because DCIS, if left untreated, may develop into invasive breast cancer. Nevertheless, there are serious side effects or sequelae to these treatments. There is, therefore, a need for efficacious breast cancer treatments.

There were an estimated 23,100 new cases of ovarian cancer in the United States in 2000. It accounts for 4% of all cancers among women and ranks second among gynecologic cancers. During 1992-1996, ovarian cancer incidence rates were significantly declining. Consequent to ovarian cancer, there were an estimated 14,000 deaths in 2000. Ovarian cancer causes more deaths than any other cancer of the female reproductive system.

Surgery, radiation therapy, and chemotherapy are treatment options for ovarian cancer. Surgery usually includes the removal of one or both ovaries, the fallopian tubes (salpingo-oophorectomy), and the uterus (hysterectomy). In some very early tumors, only the involved ovary will be removed, especially in young women who wish to have children. In advanced disease, an attempt is made to remove all intra-abdominal disease to enhance the effect of chemotherapy. There continues to be an important need for effective treatment options for ovarian cancer.

There were an estimated 28,300 new cases of pancreatic cancer in the United States in 2000. Over the past 20 years, rates of pancreatic cancer have declined in men. Rates among women have remained approximately constant but may be beginning to decline. Pancreatic cancer caused an estimated 28,200 deaths in 2000 in the United States. Over the past 20 years, there has been a slight but significant decrease in mortality rates among men (about −0.9% per year) while rates have increased slightly among women.

Surgery, radiation therapy, and chemotherapy are treatment options for pancreatic cancer. These treatment options can extend survival and/or relieve symptoms in many patients but are not likely to produce a cure for most. There is a significant need for additional therapeutic and diagnostic options for pancreatic cancer.

SUMMARY OF THE INVENTION

The present invention relates to a gene, designated 158P3D2, that has now been found to be over-expressed in the cancer(s) listed in Table 1. Northern blot expression analysis of 158P3D2 gene expression in normal tissues shows a restricted expression pattern in adult tissues. The nucleotide (FIG. 2) and amino acid (FIG. 2, and FIG. 3) sequences of 158P3D2 are provided. The tissue-related profile of 158P3D2 in normal adult tissues, combined with the over-expression observed in the tumors listed in Table 1, shows that 158P3D2 is aberrantly over-expressed in at least some cancers, and thus serves as a useful diagnostic, prophylactic, prognostic, and/or therapeutic target for cancers of the tissue(s) such as those listed in Table 1.

The invention provides polynucleotides corresponding or complementary to all or part of the 158P3D2 genes, mRNAs, and/or coding sequences, preferably in isolated form, including polynucleotides encoding 158P3D2-related proteins and fragments of 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, or more than 25 contiguous amino acids; at least 30, 35, 40, 45, 50, 55, 60, 65, 70, 80, 85, 90, 95, 100, 105, 110, 115, 120, 125, 130, 135, 140, 145, 150, 155, 160, 165, 170, 175, 180, 185, 190, 195, 200, 225, 250, 275, 300, 325, 328 or more than 328 contiguous amino acids of a 158P3D2-related protein, as well as the peptides/proteins themselves; DNA, RNA, DNA/RNA hybrids, and related molecules, polynucleotides or oligonucleotides complementary or having at least a 90% homology to the 158P3D2 genes or mRNA sequences or parts thereof, and polynucleotides or oligonucleotides that hybridize to the 158P3D2 genes, mRNAs, or to 158P3D2-encoding polynucleotides. Also provided are means for isolating cDNAs and the genes encoding 158P3D2. Recombinant DNA molecules containing 158P3D2 polynucleotides, cells transformed or transduced with such molecules, and host-vector systems for the expression of 158P3D2 gene products are also provided. The invention further provides antibodies that bind to 158P3D2 proteins and polypeptide fragments thereof, including polyclonal and monoclonal antibodies, murine and other mammalian antibodies, chimeric antibodies, humanized and fully human antibodies, and antibodies labeled with a detectable marker or therapeutic agent. In certain embodiments there is a proviso that the entire nucleic acid sequence of FIG. 2 is not encoded and/or the entire amino acid sequence of FIG. 2 is not prepared. In certain embodiments, the entire nucleic acid sequence of FIG. 2 is encoded and/or the entire amino acid sequence of FIG. 2 is prepared, either of which are in respective human unit dose forms.

The invention further provides methods for detecting the presence and status of 158P3D2 polynucleotides and proteins in various biological samples, as well as methods for identifying cells that express 158P3D2. A typical embodiment of this invention provides methods for monitoring 158P3D2 gene products in a tissue or hematology sample having or suspected of having some form of growth dysregulation such as cancer.

The invention further provides various immunogenic or therapeutic compositions and strategies for treating cancers that express 158P3D2 such as cancers of tissues listed in Table I, including therapies aimed at inhibiting the transcription, translation, processing or function of 158P3D2 as well as cancer vaccines. In one aspect, the invention provides compositions, and methods comprising them, for treating a cancer that expresses 158P3D2 in a human subject wherein the composition comprises a carrier suitable for human use and a human unit dose of one or more than one agent that inhibits the production or function of 158P3D2. Preferably, the carrier is a uniquely human carrier. In another aspect of the invention, the agent is a moiety that is immunoreactive with 158P3D2 protein. Non-limiting examples of such moieties include, but are not limited to, antibodies (such as single chain, monoclonal, polyclonal, humanized, chimeric, or human antibodies), functional equivalents thereof (whether naturally occurring or synthetic), and combinations thereof. The antibodies can be conjugated to a diagnostic or therapeutic moiety. In another aspect, the agent is a small-molecule as defined herein.

In another aspect, the agent comprises one or more than one peptide which comprises a cytotoxic T lymphocyte (CTL) epitope that binds an HLA class I molecule in a human to elicit a CTL response to 158P3D2 and/or one or more than one peptide which comprises a helper T lymphocyte (HTL) epitope which binds an HLA class II molecule in a human to elicit an HTL response. The peptides of the invention may be on the same or on one or more separate polypeptide molecules. In a further aspect of the invention, the agent comprises one or more than one nucleic acid molecule that expresses one or more than one of the CTL or HTL response stimulating peptides as described above. In yet another aspect of the invention, the one or more than one nucleic acid molecule may express a moiety that is immunologically reactive with 158P3D2 as described above. The one or more than one nucleic acid molecule may also be, or encodes, a molecule that inhibits production of 158P3D2. Non-limiting examples of such molecules include, but are not limited to, those complementary to a nucleotide sequence essential for production of 158P3D2 (e.g. antisense sequences or molecules that form a triple helix with a nucleotide double helix essential for 158P3D2 production) or a ribozyme effective to lyse 158P3D2 mRNA.

BRIEF DESCRIPTION OF THE FIGURES

FIG. 1. The 158P3D2 SSH sequence of 312 nucleotides (SEQ ID. NO:6273).

FIG. 2. The cDNA (SEQ ID NO: 6274) and amino acid sequence (SEQ ID NO:6275) of 158P3D2 variant 1 clone 158P3D2-BCP1 (also called “158P3D2 v.1” or “158P3D2 variant 1” or “158P3D2 var1”) is shown in FIG. 2A. The start methionine is underlined. The open reading frame extends from nucleic acid 849-1835 including the stop codon. The cDNA (SEQ ID NO:6276) and amino acid sequence (SEQ ID NO:6277) of 158P3D2 variant 2a (also called “158P3D2 var2a” or “158P3D2 v.2a”) is shown in FIG. 2B. The codon for the start methionine is underlined. The open reading frame extends from nucleic acid 117 to 827 including the stop codon. The cDNA (SEQ ID NO:6278) and amino acid sequence (SEQ ID NO:6279) of 158P3D2 variant 2b (also called “158P3D2 var2b” or “158P3D2 v.2b”) is shown in FIG. 2C. The codon for the start methionine is underlined. The open reading frame extends from nucleic acid 2249-2794 including the stop codon. The cDNA (SEQ ID NO:6280) and amino acid sequence (SEQ ID NO:6281) of 158P3D2 variant 3 (also called “158P3D2 var3” or “158P3D2 v.3”) is shown in FIG. 2D. The codon for the start methionine is underlined. The open reading frame extends from nucleic acid 849-1835 including the stop codon. The cDNA (SEQ ID NO:6282) and amino acid sequence (SEQ ID NO:6283) of 158P3D2 variant 4 (also called “158P3D2 var4” or “158P3D2 v.4”) is shown in FIG. 2E. The codon for the start methionine is underlined. The open reading frame extends from nucleic acid 849-1835 including the stop codon. The cDNA (SEQ ID NO:6284) and amino acid sequence (SEQ ID NO:6285) of 158P3D2 variant 5a clone 158P3D2-BCP2 (also called “158P3D2 variant 5a” or “158P3D2 var5a” or “158P3D2 v.5a”) is shown in FIG. 2F. The codon for the start methionine is underlined. The open reading frame extends from nucleic acid 849-1385 including the stop codon. The cDNA (SEQ ID NO:6286) and amino acid sequence (SEQ ID NO:6287) of 158P3D2 variant 5b clone 158P3D2-BCP2 (also called “158P3D2 variant 5b” or “158P3D2 var5b” or “158P3D2 v.5b”) is shown in FIG. 2G. The codon for the start methionine is underlined. The open reading frame extends from nucleic acid 1289-1834 including the stop codon. The cDNA (SEQ ID NO:6288) and amino acid sequence (SEQ ID NO:6289) of 158P3D2 variant 6 (also called “158P3D2 var6” or “158P3D2 v.6”) is shown in FIG. 2H. The codon for the start methionine is underlined. The open reading frame extends from nucleic acid 849-1835 including the stop codon. The cDNA (SEQ ID NO:6290) and amino acid sequence (SEQ ID NO:6291) of 158P3D2 variant 7 (also called “158P3D2 var7” or “158P3D2 v.7”) is shown in FIG. 2I. The codon for the start methionine is underlined. The open reading frame extends from nucleic acid 849-1835 including the stop codon. The cDNA (SEQ ID NO:6292) and amino acid sequence (SEQ ID NO:6293) of 158P3D2 variant 8 (also called “158P3D2 var8” or “158P3D2 v.8”) is shown in FIG. 2J. The codon for the start methionine is underlined. The open reading frame extends from nucleic acid 849-1835 including the stop codon. As used herein, a reference to 158P3D2 includes all variants thereof, including those shown in FIG. 10.

FIG. 3. Amino acid sequence of 158P3D2 var1 (SEQ ID NO:6275) is shown in FIG. 3A; it has 328 amino acids. The amino acid sequence of 158P3D2 var2a (SEQ ID NO:6277) is shown in FIG. 3B; it has 236 amino acids. The amino acid sequence of 158P3D2 var2b (SEQ ID NO:6279) is shown in FIG. 3C; it has 181 amino acids. The amino acid sequence of 158P3D2 var3 (SEQ ID NO:6281) is shown in FIG. 3D; it has 328 amino acids. The amino acid sequence of 158P3D2 var4 (SEQ ID NO:6283) is shown in FIG. 3E; it has 328 amino acids. The amino acid sequence of 158P3D2 var5a (SEQ ID NO:6285) is shown in FIG. 3F; it has 178 amino acids. The amino acid sequence of 158P3D2 var5b (SEQ ID NO:6287) is shown in FIG. 3G; it has 181 amino acids. As used herein, a reference to 158P3D2 includes all variants thereof, including those shown in FIG. 11.

FIG. 4. The nucleic acid sequence alignment of 158P3D2 var1 (SEQ. ID. No.: 6301) to fer-1-like 4 (C. elegans) (FER1L4) mRNA (SEQ. ID. No.: 6302) is shown in FIG. 4A. The amino acid sequence alignment of 158P3D2 var1 (SEQ. ID. No.: 6303) to dJ477O4.1.1 (AL121586), a novel protein similar to otoferlin and dysferlin, isoform 1 (SEQ. ID. No.: 6304) is shown in FIG. 4B. The amino acid sequence alignment of 158P3D2 v.1 (SEQ. ID. No.: 6305) with human brain otoferlin long isoform (SEQ. ID. No.: 6306) is shown in FIG. 4C. The amino acid sequence alignment of 158P3D2 v.1 (SEQ. ID. No.: 6307) with mouse otoferlin (SEQ. ID. No.: 6308) is shown in FIG. 4D. The amino acid sequence alignments of 158P3D2 protein var1 (SEQ. ID. No.: 6313), 2a (SEQ. ID. No.: 6309), 2b (SEQ. ID. No.: 6311), 3 (SEQ. ID. No.: 6315), 4 (SEQ. ID. No.: 6314), 5a (SEQ. ID. No.: 6310), and 5b (SEQ. ID. No.: 6312) are shown in FIG. 4E.

FIG. 5. Hydrophilicity amino acid profile of A) 158P3D2 var1, B) 158P3D2 var2a and C) 158P3D2 var5a, determined by computer algorithm sequence analysis using the method of Hopp and Woods (Hopp T. P., Woods K. R., 1981. Proc. Natl. Acad. Sci. U.S.A. 78:3824-3828) accessed on the Protscale website through the ExPasy molecular biology server.

FIG. 6. Hydropathicity amino acid profile of A) 158P3D2 var1, B) 158P3D2 var2a and C) 158P3D2 var5a, determined by computer algorithm sequence analysis using the method of Kyte and Doolittle (Kyte J., Doolittle R.F., 1982. J. Mol. Biol. 157:105-132) accessed on the ProtScale website through the ExPasy molecular biology server.

FIG. 7. Percent accessible residues amino acid profile of A) 158P3D2 var1, B) 158P3D2 var2a and C) 158P3D2 var5a, determined by computer algorithm sequence analysis using the method of Janin (Janin J., 1979 Nature 277:491-492) accessed on the ProtScale website through the ExPasy molecular biology server.

FIG. 8. Average flexibility amino acid profile of A) 158P3D2 var1, B) 158P3D2 var2a and C) 158P3D2 var5a, determined by computer algorithm sequence analysis using the method of Bhaskaran and Ponnuswamy (Bhaskaran R., and Ponnuswamy P. K., 1988. Int. J. Pept. Protein Res. 32:242-255) accessed on the ProtScale website through the ExPasy molecular biology server.

FIG. 9. Beta-turn amino acid profile of A) 158P3D2 var1, B) 158P3D2 var2a and C) 158P3D2 var5a, determined by computer algorithm sequence analysis using the method of Deleage and Roux (Deleage, G., Roux B. 1987 Protein Engineering 1:289-294) accessed on the ProtScale website through the ExPasy molecular biology server.

FIG. 10. Schematic display of nucleotide variants of 158P3D2. Variant 158P3D2 v.2 is an alternative transcript. Others are Single Nucleotide Polymorphism (also called “SNP”) variants, which could also occur in any alternative transcript. The numbers in “( )” underneath the box correspond to those of 158P3D2 var1. ‘-’ indicate single nucleotide deletion. Variants 158P3D2 v.3 through v.8 are variants with single nucleotide variations. The black boxes show the same sequence as 158P3D2 var1. SNPs are indicated above the box.

FIG. 11. Schematic display of protein variants of 158P3D2. Nucleotide variant 158P3D2 var2 and 158P3D2 v.5 in FIG. 10 potentially code for two different proteins, designated as variants 158P3D2 var2a and 158P3D2 var2b, 158P3D2 v.5a and 158P3D2 v.5b, respectively. Variant 158P3D2 v.5b shares the same amino acid sequence as variant 158P3D2 var2b. Variants 158P3D2 v.3 and v.4 are variants with single amino acid variations. The black boxes show the same sequence as 158P3D2 var1. The numbers in “( )” underneath the box correspond to those of 158P3D2 var1. Single amino acid differences are indicated above the box.

FIG. 12. Secondary structure prediction of 158P3D2 var1 (FIG. 12A) (SEQ. ID. No.: 6316), var2a (FIG. 12B) (SEQ. ID. No.: 6317) and var5a (FIG. 12C) (SEQ. ID. No.: 6318); and transmembrane predictions for 158P3D2 var1 (FIGS. 12D and E). The secondary structure of 158P3D2 proteins were predicted using the HNN—Hierarchial Neural Network method, accessed from the ExPasy molecular biology server. This method predicts the presence and location of alpha helices, extended strands, and random coils from the primary protein sequence. The percent of the protein in a given secondary structure is also given.

A schematic representation of the probability of existence of transmembrane regions and orientation based on the TMpred algorithm which utilizes TMBASE is shown in FIG. 12D (K. Hofmann, W. Stoffel. TMBASE—A database of membrane spanning protein segments Biol. Chem. Hoppe-Seyler 374:166, 1993). A schematic representation of the probability of the existence of transmembrane regions and the extracellular and intracellular orientation based on the TMHMM algorithm is shown in FIG. 12E (Erik L. L. Sonnhammer, Gunnar von Heijne, and Anders Krogh: A hidden Markov model for predicting transmembrane helices in protein sequences. In Proc. of Sixth Int. Conf. on Intelligent Systems for Molecular Biology, p 175-182 Ed J. Glasgow, T. Littlejohn, F. Major, R. Lathrop, D. Sankoff, and C. Sensen Menlo Park, Calif.: AAAI Press, 1998). The TMpred and TMHMM algorithms are accessed from the ExPasy molecular biology server. The results of the transmembrane prediction programs depict 158P3D2 var1 as containing 1 transmembrane domain.

FIG. 13. Exon compositions of transcript variants of 158P3D2. Variant 158P3D2 var2 is an alternative transcript. Compared with 158P3D2 var1, it has six additional exons to the 5′ end, an exon 7 longer than exon 1 of 158P3D2 var1 and an exon 10 shorter than exon 4 of 158P3D2 var1. Exons 2, 3, 5, 6 and 7 of 158P3D2 var1 are the same as exons 8, 9, 11, 12 and 13 of 158P3D2 var2, respectively. The numbers in “( )” underneath the box correspond to those of 158P3D2 var1. The black boxes show the same sequence as 158P3D2 var1. The length of the introns are not proportional.

FIG. 14. Expression of 158P3D2 by RT-PCR. First strand cDNA was prepared from vital pool 1 (liver, lung and kidney), vital pool 2 (pancreas, colon and stomach), prostate cancer metastasis to lymph node from 2 different patients, prostate cancer pool, bladder cancer pool, kidney cancer pool, colon cancer pool, lung cancer pool, ovary cancer pool, breast cancer pool, cancer metastasis pool, and pancreas cancer pool. Normalization was performed by PCR using primers to actin and GAPDH. Semi-quantitative PCR, using primers to 158P3D2, was performed at 26 and 30 cycles of amplification. Results show strong expression of 158P3D2 in bladder cancer pool, kidney cancer pool and cancer metastasis pool. Expression of 158P3D2 is also detected in colon cancer pool, lung cancer pool, ovary cancer pool, breast cancer pool, pancreas cancer pool and prostate metastases to lymph node, and vital pool 2, but not vital pool 1.

FIG. 15. Expression of 158P3D2 in normal tissues. Two multiple tissue northern blots (Clontech) both with 2 ug of mRNA/lane were probed with the 158P3D2 SSH fragment. Size standards in kilobases (kb) are indicated on the side. Results show restricted expression of an approximately 8 kb 158P3D2 transcript in normal placenta.

FIG. 16. Expression of 158P3D2 in Multiple Normal Tissues. An mRNA dot blot containing 76 different samples from human tissues was analyzed using a 158P3D2 probe. Expression was detected in placenta and stomach.

FIG. 17. Expression of 158P3D2 in Patient Cancer Specimens and Normal Tissues. RNA was extracted from a pool of three bladder cancers, as well as from normal prostate (NP), normal bladder (NB), normal kidney (NK), normal colon (NC), normal lung (NL) and normal breast (NBr). Northern blot with 10 μg of total RNA/lane was probed with 158P3D2 sequence. Size standards in kilobases (kb) are indicated on the side. The results show expression of 158P3D2 in the bladder cancer pool but not in the normal tissues tested.

FIG. 18. Expression of 158P3D2 in bladder cancer patient tissues. RNA was extracted from normal bladder (N), bladder cancer cell lines (UM-UC-3, J82, SCaBER), bladder cancer patient tumors (T) and their normal adjacent tissues (NAT). Northern blots with 10 ug of total RNA were probed with the 158P3D2 SSH fragment. Size standards in kilobases are on the side. Results show strong expression of 158P3D2 in tumor tissues. The expression observed in normal adjacent tissue (isolated from diseased tissues) but not in normal tissue, isolated from healthy donors, may indicate that these tissues are not fully normal and that 158P3D2 may be expressed in early stage tumors.

FIG. 19. 158P3D2 Expression in 293T Cells Following Transfection. 293T cells were transfected with either 158P3D2.pcDNA3.1/mychis or pcDNA3.1/mychis vector control. Forty hours later, cell lysates were collected. Samples were run on an SDS-PAGE acrylamide gel, blotted and stained with anti-his antibody. The blot was developed using the ECL chemiluminescence kit and visualized by autoradiography. Results show expression of 158P3D2 clones of 158P3D2.pcDNA3.1/mychis in the lysates of 158P3D2.pcDNA3.1/mychis transfected cells.

DETAILED DESCRIPTION OF THE INVENTION

Outline of Sections

I.) Definitions

II.) 158P3D2 Polynucleotides

-   -   II.A.) Uses of 158P3D2 Polynucleotides         -   II.A.1.) Monitoring of Genetic Abnormalities         -   II.A.2.) Antisense Embodiments         -   II.A.3.) Primers and Primer Pairs         -   II.A.4.) Isolation of 158P3D2-Encoding Nucleic Acid             Molecules         -   II.A.5.) Recombinant Nucleic Acid Molecules and Host-Vector             Systems

III.) 158P3D2-related Proteins

-   -   III.A.) Motif-bearing Protein Embodiments     -   III.B.) Expression of 158P3D2-related Proteins     -   III.C.) Modifications of 158P3D2-related Proteins     -   III.D.) Uses of 158P3D2-related Proteins

IV.) 158P3D2 Antibodies

V.) 158P3D2 Cellular Immune Responses

VI.) 158P3D2 Transgenic Animals

VII.) Methods for the Detection of 158P3D2

VIII.) Methods for Monitoring the Status of 158P3D2-related Genes and Their Products

IX.) Identification of Molecules That Interact With 158P3D2

X.) Therapeutic Methods and Compositions

-   -   X.A.) Anti-Cancer Vaccines     -   X. B.) 158P3D2 as a Target for Antibody-Based Therapy     -   X.C.) 158P3D2 as a Target for Cellular Immune Responses         -   X.C.1. Minigene Vaccines         -   X.C.2. Combinations of CTL Peptides with Helper Peptides         -   X.C.3. Combinations of CTL Peptides with T Cell Priming             Agents         -   X.C.4. Vaccine Compositions Comprising DC Pulsed with CTL             and/or HTL Peptides     -   X.D.) Adoptive Immunotherapy     -   X.E.) Administration of Vaccines for Therapeutic or Prophylactic         Purposes

XI.) Diagnostic and Prognostic Embodiments of 158P3D2.

XII.) Inhibition of 158P3D2 Protein Function

-   -   XII.A.) Inhibition of 158P3D2 With Intracellular Antibodies     -   XII.B.) Inhibition of 158P3D2 with Recombinant Proteins     -   XII.C.) Inhibition of 158P3D2 Transcription or Translation     -   XII.D.) General Considerations for Therapeutic Strategies

XIII.) KITS

I.) DEFINITIONS

Unless otherwise defined, all terms of art, notations and other scientific terms or terminology used herein are intended to have the meanings commonly understood by those of skill in the art to which this invention pertains. In some cases, terms with commonly understood meanings are defined herein for clarity and/or for ready reference, and the inclusion of such definitions herein should not necessarily be construed to represent a substantial difference over what is generally understood in the art. Many of the techniques and procedures described or referenced herein are well understood and commonly employed using conventional methodology by those skilled in the art, such as, for example, the widely utilized molecular cloning methodologies described in Sambrook et al., Molecular Cloning: A Laboratory Manual 2nd. edition (1989) Cold Spring Harbor Laboratory Press, Cold Spring Harbor, N.Y. As appropriate, procedures involving the use of commercially available kits and reagents are generally carried out in accordance with manufacturer defined protocols and/or parameters unless otherwise noted.

The terms “advanced prostate cancer”, “locally advanced prostate cancer”, “advanced disease” and “locally advanced disease” mean prostate cancers that have extended through the prostate capsule, and are meant to include stage C disease under the American Urological Association (AUA) system, stage C1-C2 disease under the Whitmore-Jewett system, and stage T3-T4 and N+ disease under the TNM (tumor, node, metastasis) system. In general, surgery is not recommended for patients with locally advanced disease, and these patients have substantially less favorable outcomes compared to patients having clinically localized (organ-confined) prostate cancer. Locally advanced disease is clinically identified by palpable evidence of induration beyond the lateral border of the prostate, or asymmetry or induration above the prostate base. Locally advanced prostate cancer is presently diagnosed pathologically following radical prostatectomy if the tumor invades or penetrates the prostatic capsule, extends into the surgical margin, or invades the seminal vesicles.

“Altering the native glycosylation pattern” is intended for purposes herein to mean deleting one or more carbohydrate moieties found in native sequence 158P3D2 (either by removing the underlying glycosylation site or by deleting the glycosylation by chemical and/or enzymatic means), and/or adding one or more glycosylation sites that are not present in the native sequence 158P3D2. In addition, the phrase includes qualitative changes in the glycosylation of the native proteins, involving a change in the nature and proportions of the various carbohydrate moieties present.

The term “analog” refers to a molecule which is structurally similar or shares similar or corresponding attributes with another molecule (e.g. a 158P3D2-related protein). For example an analog of a 158P3D2 protein can be specifically bound by an antibody or T cell that specifically binds to 158P3D2.

The term “antibody” is used in the broadest sense. Therefore an “antibody” can be naturally occurring or man-made such as monoclonal antibodies produced by conventional hybridoma technology. Anti-158P3D2 antibodies comprise monoclonal and polyclonal antibodies as well as fragments containing the antigen-binding domain and/or one or more complementarity determining regions of these antibodies.

An “antibody fragment” is defined as at least a portion of the variable region of the immunoglobulin molecule that binds to its target, i.e., the antigen-binding region. In one embodiment it specifically covers single anti-158P3D2 antibodies and clones thereof (including agonist, antagonist and neutralizing antibodies) and anti-158P3D2 antibody compositions with polyepitopic specificity.

The term “codon optimized sequences” refers to nucleotide sequences that have been optimized for a particular host species by replacing any codons having a usage frequency of less than about 20%. Nucleotide sequences that have been optimized for expression in a given host species by elimination of spurious polyadenylation sequences, elimination of exon/intron splicing signals, elimination of transposon-like repeats and/or optimization of GC content in addition to codon optimization are referred to herein as an “expression enhanced sequences.”

The term “cytotoxic agent” refers to a substance that inhibits or prevents the expression activity of cells, function of cells and/or causes destruction of cells. The term is intended to include radioactive isotopes chemotherapeutic agents, and toxins such as small molecule toxins or enzymatically active toxins of bacterial, fungal, plant or animal origin, including fragments and/or variants thereof. Examples of cytotoxic agents include, but are not limited to maytansinoids, yttrium, bismuth, ricin, ricin A-chain, doxorubicin, daunorubicin, taxol, ethidium bromide, mitomycin, etoposide, tenoposide, vincristine, vinblastine, colchicine, dihydroxy anthracin dione, actinomycin, diphtheria toxin, Pseudomonas exotoxin (PE) A, PE40, abrin, abrin A chain, modeccin A chain, alpha-sarcin, gelonin, mitogellin, retstrictocin, phenomycin, enomycin, curicin, crotin, calicheamicin, sapaonaria officinalis inhibitor, and glucocorticoid and other chemotherapeutic agents, as well as radioisotopes such as At²¹¹, I¹³¹, I¹²⁵, Y⁹⁰, Re¹⁸⁶, Re¹⁸⁸, Sm¹⁵³, Bi²¹², P³² and radioactive isotopes of Lu. Antibodies may also be conjugated to an anti-cancer pro-drug activating enzyme capable of converting the pro-drug to its active form.

The term “homolog” refers to a molecule which exhibits homology to another molecule, by for example, having sequences of chemical residues that are the same or similar at corresponding positions.

“Human Leukocyte Antigen” or “HLA” is a human class I or class II Major Histocompatibility Complex (MHC) protein (see, e.g., Stites, et al., IMMUNOLOGY, 8^(TH) ED., Lange Publishing, Los Altos, Calif. (1994).

The terms “hybridize”, “hybridizing”, “hybridizes” and the like, used in the context of polynucleotides, are meant to refer to conventional hybridization conditions, preferably such as hybridization in 50% formamide/6×SSC/0.1% SDS/100 μg/ml ssDNA, in which temperatures for hybridization are above 37 degrees C. and temperatures for washing in 0.1×SSC/0.1% SDS are above 55 degrees C.

The phrases “isolated” or “biologically pure” refer to material which is substantially or essentially free from components which normally accompany the material as it is found in its native state. Thus, isolated peptides in accordance with the invention preferably do not contain materials normally associated with the peptides in their in situ environment. For example, a polynucleotide is said to be “isolated” when it is substantially separated from contaminant polynucleotides that correspond or are complementary to genes other than the 158P3D2 genes or that encode polypeptides other than 158P3D2 gene product or fragments thereof. A skilled artisan can readily employ nucleic acid isolation procedures to obtain an isolated 158P3D2 polynucleotide. A protein is said to be “isolated,” for example, when physical, mechanical or chemical methods are employed to remove the 158P3D2 proteins from cellular constituents that are normally associated with the protein. A skilled artisan can readily employ standard purification methods to obtain an isolated 158P3D2 protein. Alternatively, an isolated protein can be prepared by chemical means.

The term “mammal” refers to any organism classified as a mammal, including mice, rats, rabbits, dogs, cats, cows, horses and humans. In one embodiment of the invention, the mammal is a mouse. In another embodiment of the invention, the mammal is a human.

The terms “metastatic prostate cancer” and “metastatic disease” mean prostate cancers that have spread to regional lymph nodes or to distant sites, and are meant to include stage D disease under the AUA system and stage T×N×M+ under the TNM system. As is the case with locally advanced prostate cancer, surgery is generally not indicated for patients with metastatic disease, and hormonal (androgen ablation) therapy is a preferred treatment modality. Patients with metastatic prostate cancer eventually develop an androgen-refractory state within 12 to 18 months of treatment initiation. Approximately half of these androgen-refractory patients die within 6 months after developing that status. The most common site for prostate cancer metastasis is bone. Prostate cancer bone metastases are often osteoblastic rather than osteolytic (i.e., resulting in net bone formation). Bone metastases are found most frequently in the spine, followed by the femur, pelvis, rib cage, skull and humerus. Other common sites for metastasis include lymph nodes, lung, liver and brain. Metastatic prostate cancer is typically diagnosed by open or laparoscopic pelvic lymphadenectomy, whole body radionuclide scans, skeletal radiography, and/or bone lesion biopsy.

The term “monoclonal antibody” refers to an antibody obtained from a population of substantially homogeneous antibodies, i.e., the antibodies comprising the population are identical except for possible naturally occurring mutations that are present in minor amounts.

A “motif”, as in biological motif of an 158P3D2-related protein, refers to any pattern of amino acids forming part of the primary sequence of a protein, that is associated with a particular function (e.g. protein-protein interaction, protein-DNA interaction, etc) or modification (e.g. that is phosphorylated, glycosylated or amidated), or localization (e.g. secretory sequence, nuclear localization sequence, etc.) or a sequence that is correlated with being immunogenic, either humorally or cellularly. A motif can be either contiguous or capable of being aligned to certain positions that are generally correlated with a certain function or property. In the context of HLA motifs, “motif” refers to the pattern of residues in a peptide of defined length, usually a peptide of from about 8 to about 13 amino acids for a class I HLA motif and from about 6 to about 25 amino acids for a class II HLA motif, which is recognized by a particular HLA molecule. Peptide motifs for HLA binding are typically different for each protein encoded by each human HLA allele and differ in the pattern of the primary and secondary anchor residues.

A “pharmaceutical excipient” comprises a material such as an adjuvant, a carrier, pH-adjusting and buffering agents, tonicity adjusting agents, wetting agents, preservative, and the like.

“Pharmaceutically acceptable” refers to a non-toxic, inert, and/or composition that is physiologically compatible with humans or other mammals.

The term “polynucleotide” means a polymeric form of nucleotides of at least 10 bases or base pairs in length, either ribonucleotides or deoxynucleotides or a modified form of either type of nucleotide, and is meant to include single and double stranded forms of DNA and/or RNA. In the art, this term if often used interchangeably with “oligonucleotide”. A polynucleotide can comprise a nucleotide sequence disclosed herein wherein thymidine (T), as shown for example in FIG. 2, can also be uracil (U); this definition pertains to the differences between the chemical structures of DNA and RNA, in particular the observation that one of the four major bases in RNA is uracil (U) instead of thymidine (T).

The term “polypeptide” means a polymer of at least about 4, 5, 6, 7, or 8 amino acids. Throughout the specification, standard three letter or single letter designations for amino acids are used. In the art, this term is often used interchangeably with “peptide” or “protein”.

An HLA “primary anchor residue” is an amino acid at a specific position along a peptide sequence which is understood to provide a contact point between the immunogenic peptide and the HLA molecule. One to three, usually two, primary anchor residues within a peptide of defined length generally defines a “motif” for an immunogenic peptide. These residues are understood to fit in close contact with peptide binding groove of an HLA molecule, with their side chains buried in specific pockets of the binding groove. In one embodiment, for example, the primary anchor residues for an HLA class 1 molecule are located at position 2 (from the amino terminal position) and at the carboxyl terminal position of a 8, 9, 10, 11, or 12 residue peptide epitope in accordance with the invention. In another embodiment, for example, the primary anchor residues of a peptide that will bind an HLA class II molecule are spaced relative to each other, rather than to the termini of a peptide, where the peptide is generally of at least 9 amino acids in length. The primary anchor positions for each motif and supermotif are set forth in Table IV. For example, analog peptides can be created by altering the presence or absence of particular residues in the primary and/or secondary anchor positions shown in Table IV. Such analogs are used to modulate the binding affinity and/or population coverage of a peptide comprising a particular HLA motif or supermotif.

A “recombinant” DNA or RNA molecule is a DNA or RNA molecule that has been subjected to molecular manipulation in vitro.

Non-limiting examples of small molecules include compounds that bind or interact with 158P3D2, ligands including hormones, neuropeptides, chemokines, odorants, phospholipids, and functional equivalents thereof that bind and preferably inhibit 158P3D2 protein function. Such non-limiting small molecules preferably have a molecular weight of less than about 10 kDa, more preferably below about 9, about 8, about 7, about 6, about 5 or about 4 kDa. In certain embodiments, small molecules physically associate with, or bind, 158P3D2 protein; are not found in naturally occurring metabolic pathways; and/or are more soluble in aqueous than non-aqueous solutions

“Stringency” of hybridization reactions is readily determinable by one of ordinary skill in the art, and generally is an empirical calculation dependent upon probe length, washing temperature, and salt concentration. In general, longer probes require higher temperatures for proper annealing, while shorter probes need lower temperatures. Hybridization generally depends on the ability of denatured nucleic acid sequences to reanneal when complementary strands are present in an environment below their melting temperature. The higher the degree of desired homology between the probe and hybridizable sequence, the higher the relative temperature that can be used. As a result, it follows that higher relative temperatures would tend to make the reaction conditions more stringent, while lower temperatures less so. For additional details and explanation of stringency of hybridization reactions, see Ausubel et al., Current Protocols in Molecular Biology, Wiley Interscience Publishers, (1995).

“Stringent conditions” or “high stringency conditions”, as defined herein, are identified by, but not limited to, those that: (1) employ low ionic strength and high temperature for washing, for example 0.015 M sodium chloride/0.0015 M sodium citrate/0.1% sodium dodecyl sulfate at 50° C.; (2) employ during hybridization a denaturing agent, such as formamide, for example, 50% (v/v) formamide with 0.1% bovine serum albumin/0.1% Ficoll/0.1% polyvinylpyrrolidone/50 mM sodium phosphate buffer at pH 6.5 with 750 mM sodium chloride, 75 mM sodium citrate at 42° C.; or (3) employ 50% formamide, 5×SSC (0.75 M NaCl, 0.075 M sodium citrate), 50 mM sodium phosphate (pH 6.8), 0.1% sodium pyrophosphate, 5×Denhardt's solution, sonicated salmon sperm DNA (50 μg/ml), 0.1% SDS, and 10% dextran sulfate at 42° C., with washes at 42° C. in 0.2×SSC (sodium chloride/sodium. citrate) and 50% formamide at 55° C., followed by a high-stringency wash consisting of 0.1×SSC containing EDTA at 55° C. “Moderately stringent conditions” are described by, but not limited to, those in Sambrook et al., Molecular Cloning: A Laboratory Manual, New York: Cold Spring Harbor Press, 1989, and include the use of washing solution and hybridization conditions (e.g., temperature, ionic strength and % SDS) less stringent than those described above. An example of moderately stringent conditions is overnight incubation at 37° C. in a solution comprising: 20% formamide, 5×SSC (150 mM NaCl, 15 mM trisodium citrate), 50 mM sodium phosphate (pH 7.6), 5×Denhardt's solution, 10% dextran sulfate, and 20 mg/mL denatured sheared salmon sperm DNA, followed by washing the filters in 1×SSC at about 37-50° C. The skilled artisan will recognize how to adjust the temperature, ionic strength, etc. as necessary to accommodate factors such as probe length and the like.

An HLA “supermotif” is a peptide binding specificity shared by HLA molecules encoded by two or more HLA alleles.

As used herein “to treat” or “therapeutic” and grammatically related terms, refer to any improvement of any consequence of disease, such as prolonged survival, less morbidity, and/or a lessening of side effects which are the byproducts of an alternative therapeutic modality; full eradication of disease is not required.

A “transgenic animal” (e.g., a mouse or rat) is an animal having cells that contain a transgene, which transgene was introduced into the animal or an ancestor of the animal at a prenatal, e.g., an embryonic stage.

A “transgene” is a DNA that is integrated into the genome of a cell from which a transgenic animal develops.

As used herein, an HLA or cellular immune response “vaccine” is a composition that contains or encodes one or more peptides of the invention. There are numerous embodiments of such vaccines, such as a cocktail of one or more individual peptides; one or more peptides of the invention comprised by a polyepitopic peptide; or nucleic acids that encode such individual peptides or polypeptides, e.g., a minigene that encodes a polyepitopic peptide. The “one or more peptides” can include any whole unit integer from 1-328 or more, e.g., at least 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50, 55, 60, 65, 70, 75, 80, 85, 90, 95, 100, 105, 110, 115, 120, 125, 130, 135, 140, 145, 150, 155, 160, 165, 170, 175, 180, 185, 190, 195, 200, 225, 250, 275, 300, 325, or 328 or more peptides of the invention. The peptides or polypeptides can optionally be modified, such as by lipidation, addition of targeting or other sequences. HLA class I peptides of the invention can be admixed with, or linked to, HLA class II peptides, to facilitate activation of both cytotoxic T lymphocytes and helper T lymphocytes. HLA vaccines can also comprise peptide-pulsed antigen presenting cells, e.g., dendritic cells.

The term “variant” refers to a molecule that exhibits a variation from a described type or norm, such as a protein that has one or more different amino acid residues in the corresponding position(s) of a specifically described protein (e.g. the 158P3D2 protein shown in FIG. 2 or FIG. 3. An analog is an example of a variant protein. Splice isoforms and SNPs are further examples of variants.

The “158P3D2-related proteins” of the invention include those specifically identified herein, as well as allelic variants, conservative substitution variants, analogs and homologs that can be isolated/generated and characterized without undue experimentation following the methods outlined herein or readily available in the art. Fusion proteins that combine parts of different 158P3D2 proteins or fragments thereof, as well as fusion proteins of a 158P3D2 protein and a heterologous polypeptide are also included. Such 158P3D2 proteins are collectively referred to as the 158P3D2-related proteins, the proteins of the invention, or 158P3D2. The term “158P3D2-related protein” refers to a polypeptide fragment or an 158P3D2 protein sequence of 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, or more than 25 amino acids; or, at least 30, 35, 40, 45, 50, 55, 60, 65, 70, 80, 85, 90, 95, 100, 105, 110, 115, 120, 125, 130, 135, 140, 145, 150, 155, 160, 165, 170, 175, 180, 185, 190, 195, 200, 225, 250, 275, 300, 325, 328 or more than 328 amino acids.

II.) 158P3D2 POLYNUCLEOTIDES

One aspect of the invention provides polynucleotides corresponding or complementary to all or part of an 158P3D2 gene, mRNA, and/or coding sequence, preferably in isolated form, including polynucleotides encoding an 158P3D2-related protein and fragments thereof, DNA, RNA, DNA/RNA hybrid, and related molecules, polynucleotides or oligonucleotides complementary to an 158P3D2 gene or mRNA sequence or a part thereof, and polynucleotides or oligonucleotides that hybridize to an 158P3D2 gene, mRNA, or to an 158P3D2 encoding polynucleotide (collectively, “158P3D2 polynucleotides”). In all instances when referred to in this section, T can also be U in FIG. 2.

Embodiments of a 158P3D2 polynucleotide include: a 158P3D2 polynucleotide having the sequence shown in FIG. 2, the nucleotide sequence of 158P3D2 as shown in FIG. 2 wherein T is U; at least 10 contiguous nucleotides of a polynucleotide having the sequence as shown in FIG. 2; or, at least 10 contiguous nucleotides of a polynucleotide having the sequence as shown in FIG. 2 where T is U. For example, embodiments of 158P3D2 nucleotides comprise, without limitation:

-   -   (I) a polynucleotide comprising, consisting essentially of, or         consisting of a sequence as shown in FIG. 2A, wherein T can also         be U;     -   (II) a polynucleotide comprising, consisting essentially of, or         consisting of the sequence as shown in FIG. 2A, from nucleotide         residue number 849 through nucleotide residue number 1835,         including the stop codon, wherein T can also be U;     -   (III) a polynucleotide comprising, consisting essentially of, or         consisting of the sequence as shown in FIG. 2B, from nucleotide         residue number 117 through nucleotide residue number 827,         including the stop codon, wherein T can also be U;     -   (IV) a polynucleotide comprising, consisting essentially of, or         consisting of the sequence as shown in FIG. 2C, from nucleotide         residue number 2249 through nucleotide residue number 2794,         including the a stop codon, wherein T can also be U;     -   (V) a polynucleotide comprising, consisting essentially of, or         consisting of the sequence as shown in FIG. 2D, from nucleotide         residue number 849 through nucleotide residue number 1835,         including the stop codon, wherein T can also be U;     -   (VI) a polynucleotide comprising, consisting essentially of, or         consisting of the sequence as shown in FIG. 2E, from nucleotide         residue number 849 through nucleotide residue number 1835,         including the stop codon, wherein T can also be U;     -   (VII) a polynucleotide comprising, consisting essentially of, or         consisting of the sequence as shown in FIG. 2F, from nucleotide         residue number 849 through nucleotide residue number 1385,         including the stop codon, wherein T can also be U;     -   (VIII) a polynucleotide comprising, consisting essentially of,         or consisting of the sequence as shown in FIG. 2G, from         nucleotide residue number 1289 through nucleotide residue number         1834, including the stop codon, wherein T can also be U;     -   (IX) a polynucleotide comprising, consisting essentially of, or         consisting of the sequence as shown in FIG. 2H, from nucleotide         residue number 849 through nucleotide residue number 1835,         including the stop codon, wherein T can also be U;     -   (X) a polynucleotide comprising, consisting essentially of, or         consisting of the sequence as shown in FIG. 2I, from nucleotide         residue number 849 through nucleotide residue number 1835,         including the stop codon, wherein T can also be U;     -   (XI) a polynucleotide comprising, consisting essentially of, or         consisting of the sequence as shown in FIG. 2J, from nucleotide         residue number 849 through nucleotide residue number 1835,         including the stop codon, wherein T can also be U;     -   (XII) a polynucleotide that encodes an 158P3D2-related protein         that is at least 90% homologous to an entire amino acid sequence         shown in FIG. 2A-I;     -   (XIII) a polynucleotide that encodes an 158P3D2-related protein         that is at least 90% identical to an entire amino acid sequence         shown in FIG. 2A-I;     -   (XIV) a polynucleotide that encodes at least one peptide set         forth in Tables V-XIX;     -   (XV) a polynucleotide that encodes a peptide region of at least         S amino acids of a peptide of FIG. 3A in any whole number         increment up to 328 that includes an amino acid position having         a value greater than 0.5 in the Hydrophilicity profile of FIG.         5A; or of FIG. 3B in any whole number increment up to 236 that         includes an amino acid position having a value greater than 0.5         in the Hydrophilicity profile of FIG. 5B; or FIG. 3F in any         whole number increment up to 178 that includes an amino acid         position having a value greater than 0.5 in the Hydrophilicity         profile of FIG. 5C;     -   (XVI) a polynucleotide that encodes a peptide region of at least         5 amino acids of a peptide of FIG. 3A in any whole number         increment up to 328 that includes an amino acid position having         a value less than 0.5 in the Hydropathicity profile of FIG. 6A;         or of FIG. 3B in any whole number increment up to 236, that         includes an amino acid position having a value less than 0.5 in         the Hydropathicity profile of FIG. 6B; or FIG. 3F in any whole         number increment up to 178 that includes an amino acid position         having a value greater than 0.5 in the Hydropathicity profile of         FIG. 6C;     -   (XVII) a polynucleotide that encodes a peptide region of at         least 5 amino acids of a peptide of FIG. 3A in any whole number         increment up to 328 that includes an amino acid position having         a value greater than 0.5 in the Percent Accessible Residues         profile of FIG. 7A; or of FIG. 3B in any whole number increment         up to 236, that includes an amino acid position having a value         greater than 0.5 in the Percent Accessible Residues profile of         FIG. 7B; or FIG. 3F in any whole number increment up to 178 that         includes an amino acid position having a value greater than 0.5         in the Percent Accessible Residues profile of FIG. 7C;     -   (XVIII) a polynucleotide that encodes a peptide region of at         least S amino acids of a peptide of FIG. 3A in any whole number         increment up to 328 that includes an amino acid position having         a value greater than 0.5 in the Average Flexibility profile on         FIG. 8A; or of FIG. 3B in any whole number increment up to 236,         that includes an amino acid position having a value greater than         0.5 in the Average Flexibility profile on FIG. 8B; or FIG. 3F in         any whole number increment up to 178 that includes an amino acid         position having a value greater than 0.5 in the Average         Flexibility profile of FIG. 8C;     -   (XIX) a polynucleotide that encodes a peptide region of at least         5 amino acids of a peptide of FIG. 3A in any whole number         increment up to 328 that includes an amino acid position having         a value greater than 0.5 in the Beta-turn profile of FIG. 9A; or         of FIG. 3B in any whole number increment up to 236, that         includes an amino acid position having a value greater than 0.5         in the Beta-turn profile of FIG. 9B; or FIG. 3F in any whole         number increment up to 178 that includes an amino acid position         having a value greater than 0.5 in the Beta-turn profile of FIG.         9C;     -   (XX) a polynucleotide that is fully complementary to a         polynucleotide of any one of (I)-(XIX).     -   (XXI) a peptide that is encoded by any of (I)-(XX); and     -   (XXII) a polynucleotide of any of (I)-(XX) or peptide of (XXI)         together with a pharmaceutical excipient and/or in a human unit         dose form.

As used herein, a range is understood to specifically disclose all whole unit positions thereof.

Typical embodiments of the invention disclosed herein include 158P3D2 polynucleotides that encode specific portions of 158P3D2 mRNA sequences (and those which are complementary to such sequences) such as those that encode the proteins and/or fragments thereof, for example:

(a) 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 30, 35, 40, 45, 50, 55, 60, 65, 70, 75, 80, 85, 90, 95, 100, 105, 110, 115, 120, 125, 130, 135, 140, 145, 150, 155, 160, 165, 170, 175, 180, 185, 190, 195, 200, 225, 250, 275, 300, 325, 328 or more than 328 contiguous amino acids of 158P3D2.

For example, representative embodiments of the invention disclosed herein include: polynucleotides and their encoded peptides themselves encoding about amino acid 1 to about amino acid 10 of the 158P3D2 protein shown in FIG. 2 or FIG. 3, polynucleotides encoding about amino acid 10 to about amino acid 20 of the 158P3D2 protein shown in FIG. 2 or FIG. 3, polynucleotides encoding about amino acid 20 to about amino acid 30 of the 158P3D2 protein shown in FIG. 2 or FIG. 3, polynucleotides encoding about amino acid 30 to about amino acid 40 of the 158P3D2 protein shown in FIG. 2 or FIG. 3, polynucleotides encoding about amino acid 40 to about amino acid 50 of the 158P3D2 protein shown in FIG. 2 or FIG. 3, polynucleotides encoding about amino acid 50 to about amino acid 60 of the 158P3D2 protein shown in FIG. 2 or FIG. 3, polynucleotides encoding about amino acid 60 to about amino acid 70 of the 158P3D2 protein shown in FIG. 2 or FIG. 3, polynucleotides encoding about amino acid 70 to about amino acid 80 of the 158P3D2 protein shown in FIG. 2 or FIG. 3, polynucleotides encoding about amino acid 80 to about amino acid 90 of the 158P3D2 protein shown in FIG. 2 or FIG. 3, polynucleotides encoding about amino acid 90 to about amino acid 100 of the 158P3D2 protein shown in FIG. 2 or FIG. 3, in increments of about 10 amino acids, ending at the carboxyl terminal amino acid set forth in FIG. 2 or FIG. 3. Accordingly polynucleotides encoding portions of the amino acid sequence (of about 10 amino acids), of amino acids 100 through the carboxyl terminal amino acid of the 158P3D2 protein are embodiments of the invention. Wherein it is understood that each particular amino acid position discloses that position plus or minus five amino acid residues.

Polynucleotides encoding relatively long portions of a 158P3D2 protein are also within the scope of the invention. For example, polynucleotides encoding from about amino acid 1 (or 20 or 30 or 40 etc.) to about amino acid 20, (or 30, or 40 or 50 etc.) of the 158P3D2 protein shown in FIG. 2 or FIG. 3 can be generated by a variety of techniques well known in the art. These polynucleotide fragments can include any portion of the 158P3D2 sequence as shown in FIG. 2.

Additional illustrative embodiments of the invention disclosed herein include 158P3D2 polynucleotide fragments encoding one or more of the biological motifs contained within a 158P3D2 protein sequence, including one or more of the motif-bearing subsequences of a 158P3D2 protein set forth in Tables V-XIX. In another embodiment, typical polynucleotide fragments of the invention encode one or more of the regions of 158P3D2 protein or variant that exhibit homology to a known molecule. In another embodiment of the invention, typical polynucleotide fragments can encode one or more of the 158P3D2 protein or variant N-glycosylation sites, cAMP and cGMP-dependent protein kinase phosphorylation sites, casein kinase II phosphorylation sites or N-myristoylation site and amidation sites.

II.A.) Uses of 158P3D2 Polynucleotides II.A.1.) Monitoring of Genetic Abnormalities

The polynucleotides of the preceding paragraphs have a number of different specific uses. The human 158P3D2 gene maps to the chromosomal location set forth in Example 3. For example, because the 158P3D2 gene maps to this chromosome, polynucleotides that encode different regions of the 158P3D2 proteins are used to characterize cytogenetic abnormalities of this chromosomal locale, such as abnormalities that are identified as being associated with various cancers. In certain genes, a variety of chromosomal abnormalities including rearrangements have been identified as frequent cytogenetic abnormalities in a number of different cancers (see e.g. Krajinovic et al., Mutat. Res. 382(3-4): 81-83 (1998); Johansson et al., Blood 86(10): 3905-3914 (1995) and Finger et al., P.N.A.S. 85(23): 9158-9162 (1988)). Thus, polynucleotides encoding specific regions of the 158P3D2 proteins provide new tools that can be used to delineate, with greater precision than previously possible, cytogenetic abnormalities in the chromosomal region that encodes 158P3D2 that may contribute to the malignant phenotype. In this context, these polynucleotides satisfy a need in the art for expanding the sensitivity of chromosomal screening in order to identify more subtle and less common chromosomal abnormalities (see e.g. Evans et al., Am. J. Obstet. Gynecol 171(4): 1055-1057 (1994)).

Furthermore, as 158P3D2 was shown to be highly expressed in bladder and other cancers, 158P3D2 polynucleotides are used in methods assessing the status of 158P3D2 gene products in normal versus cancerous tissues. Typically, polynucleotides that encode specific regions of the 158P3D2 proteins are used to assess the presence of perturbations (such as deletions, insertions, point mutations, or alterations resulting in a loss of an antigen etc.) in specific regions of the 158P3D2 gene, such as regions containing one or more motifs. Exemplary assays include both RT-PCR assays as well as single-strand conformation polymorphism (SSCP) analysis (see, e.g., Marrogi et al., J. Cutan. Pathol. 26(8): 369-378 (1999), both of which utilize polynucleotides encoding specific regions of a protein to examine these regions within the protein.

II.A.2.) Antisense Embodiments

Other specifically contemplated nucleic acid related embodiments of the invention disclosed herein are genomic DNA, cDNAs, ribozymes, and antisense molecules, as well as nucleic acid molecules based on an alternative backbone, or including alternative bases, whether derived from natural sources or synthesized, and include molecules capable of inhibiting the RNA or protein expression of 158P3D2. For example, antisense molecules can be RNAs or other molecules, including peptide nucleic acids (PNAs) or non-nucleic acid molecules such as phosphorothioate derivatives, that specifically bind DNA or RNA in a base pair-dependent manner. A skilled artisan can readily obtain these classes of nucleic acid molecules using the 158P3D2 polynucleotides and polynucleotide sequences disclosed herein.

Antisense technology entails the administration of exogenous oligonucleotides that bind to a target polynucleotide located within the cells. The term “antisense” refers to the fact that such oligonucleotides are complementary to their intracellular targets, e.g., 158P3D2. See for example, Jack Cohen, Oligodeoxynucleotides, Antisense Inhibitors of Gene Expression, CRC Press, 1989; and Synthesis 1:1-5 (1988). The 158P3D2 antisense oligonucleotides of the present invention include derivatives such as S-oligonucleotides (phosphorothioate derivatives or S-oligos, see, Jack Cohen, supra), which exhibit enhanced cancer cell growth inhibitory action. S-oligos (nucleoside phosphorothioates) are isoelectronic analogs of an oligonucleotide (O-oligo) in which a nonbridging oxygen atom of the phosphate group is replaced by a sulfur atom. The S-oligos of the present invention can be prepared by treatment of the corresponding O-oligos with 3H-1,2-benzodithiol-3-one-1,1-dioxide, which is a sulfur transfer reagent. See, e.g., Iyer, R. P. et al., J. Org. Chem. 55:4693-4698 (1990); and Iyer, R. P. et al., J. Am. Chem. Soc. 112:1253-1254 (1990). Additional 158P3D2 antisense oligonucleotides of the present invention include morpholino antisense oligonucleotides known in the art (see, e.g., Partridge et al., 1996, Antisense & Nucleic Acid Drug Development 6: 169-175).

The 158P3D2 antisense oligonucleotides of the present invention typically can be RNA or DNA that is complementary to and stably hybridizes with the first 100 5′ codons or last 100 3′ codons of a 158P3D2 genomic sequence or the corresponding mRNA. Absolute complementarity is not required, although high degrees of complementarity are preferred. Use of an oligonucleotide complementary to this region allows for the selective hybridization to 158P3D2 mRNA and not to mRNA specifying other regulatory subunits of protein kinase. In one embodiment, 158P3D2 antisense oligonucleotides of the present invention are 15 to 30-mer fragments of the antisense DNA molecule that have a sequence that hybridizes to 158P3D2 mRNA. Optionally, 158P3D2 antisense oligonucleotide is a 30-mer oligonucleotide that is complementary to a region in the first 10 5′ codons or last 10 3′ codons of 158P3D2. Alternatively, the antisense molecules are modified to employ ribozymes in the inhibition of 158P3D2 expression, see, e.g., L. A. Couture & D. T. Stinchcomb; Trends Genet 12: 510-515 (1996).

II.A.3.) Primers and Primer Pairs

Further specific embodiments of this nucleotides of the invention include primers and primer pairs, which allow the specific amplification of polynucleotides of the invention or of any specific parts thereof, and probes that selectively or specifically hybridize to nucleic acid molecules of the invention or to any part thereof. Probes can be labeled with a detectable marker, such as, for example, a radioisotope, fluorescent compound, bioluminescent compound, a chemiluminescent compound, metal chelator or enzyme. Such probes and primers are used to detect the presence of a 158P3D2 polynucleotide in a sample and as a means for detecting a cell expressing a 158P3D2 protein.

Examples of such probes include polypeptides comprising all or part of the human 158P3D2 cDNA sequence shown in FIG. 2. Examples of primer pairs capable of specifically amplifying 158P3D2 mRNAs are also described in the Examples. As will be understood by the skilled artisan, a great many different primers and probes can be prepared based on the sequences provided herein and used effectively to amplify and/or detect a 158P3D2 mRNA.

The 158P3D2 polynucleotides of the invention are useful for a variety of purposes, including but not limited to their use as probes and primers for the amplification and/or detection of the 158P3D2 gene(s), mRNA(s), or fragments thereof; as reagents for the diagnosis and/or prognosis of prostate cancer and other cancers; as coding sequences capable of directing the expression of 158P3D2 polypeptides; as tools for modulating or inhibiting the expression of the 158P3D2 gene(s) and/or translation of the 158P3D2 transcript(s); and as therapeutic agents.

The present invention includes the use of any probe as described herein to identify and isolate a 158P3D2 or 158P3D2 related nucleic acid sequence from a naturally occurring source, such as humans or other mammals, as well as the isolated nucleic acid sequence per se, which would comprise all or most of the sequences found in the probe used.

II.A.4.) Isolation of 158P3D2-Encoding Nucleic Acid Molecules

The 158P3D2 cDNA sequences described herein enable the isolation of other polynucleotides encoding 158P3D2 gene product(s), as well as the isolation of polynucleotides encoding 158P3D2 gene product homologs, alternatively spliced isoforms, allelic variants, and mutant forms of a 158P3D2 gene product as well as polynucleotides that encode analogs of 158P3D2-related proteins. Various molecular cloning methods that can be employed to isolate full length cDNAs encoding an 158P3D2 gene are well known (see, for example, Sambrook, J. et al., Molecular Cloning: A Laboratory Manual, 2d edition, Cold Spring Harbor Press, New York, 1989; Current Protocols in Molecular Biology. Ausubel et al., Eds., Wiley and Sons, 1995). For example, lambda phage cloning methodologies can be conveniently employed, using commercially available cloning systems (e.g., Lambda ZAP Express, Stratagene). Phage clones containing 158P3D2 gene cDNAs can be identified by probing with a labeled 158P3D2 cDNA or a fragment thereof. For example, in one embodiment, a 158P3D2 cDNA (e.g., FIG. 2) or a portion thereof can be synthesized and used as a probe to retrieve overlapping and full-length cDNAs corresponding to a 158P3D2 gene. A 158P3D2 gene itself can be isolated by screening genomic DNA libraries, bacterial artificial chromosome libraries (BACs), yeast artificial chromosome libraries (YACs), and the like, with 158P3D2 DNA probes or primers.

II.A.5.) Recombinant Nucleic Acid Molecules and Host-Vector Systems

The invention also provides recombinant DNA or RNA molecules containing an 158P3D2 polynucleotide, a fragment, analog or homologue thereof, including but not limited to phages, plasmids, phagemids, cosmids, YACs, BACs, as well as various viral and non-viral vectors well known in the art, and cells transformed or transfected with such recombinant DNA or RNA molecules. Methods for generating such molecules are well known (see, for example, Sambrook et al., 1989, supra).

The invention further provides a host-vector system comprising a recombinant DNA molecule containing a 158P3D2 polynucleotide, fragment, analog or homologue thereof within a suitable prokaryotic or eukaryotic host cell. Examples of suitable eukaryotic host cells include a yeast cell, a plant cell, or an animal cell, such as a mammalian cell or an insect cell (e.g., a baculovirus-infectible cell such as an Sf9 or HighFive cell). Examples of suitable mammalian cells include various prostate cancer cell lines such as DU145 and TsuPr1, other transfectable or transducible prostate cancer cell lines, primary cells (PrEC), as well as a number of mammalian cells routinely used for the expression of recombinant proteins (e.g., COS, CHO, 293, 293T cells). More particularly, a polynucleotide comprising the coding sequence of 158P3D2 or a fragment, analog or homolog thereof can be used to generate 158P3D2 proteins or fragments thereof using any number of host-vector systems routinely used and widely known in the art.

A wide range of host-vector systems suitable for the expression of 158P3D2 proteins or fragments thereof are available, see for example, Sambrook et al., 1989, supra; Current Protocols in Molecular Biology, 1995, supra). Preferred vectors for mammalian expression include but are not limited to pcDNA 3.1 myc-His-tag (Invitrogen) and the retroviral vector pSRαtkneo (Muller et al., 1991, MCB 11:1785). Using these expression vectors, 158P3D2 can be expressed in several prostate cancer and non-prostate cell lines, including for example 293, 293T, rat-1, NIH 3T3 and TsuPr1. The host-vector systems of the invention are useful for the production of a 158P3D2 protein or fragment thereof. Such host-vector systems can be employed to study the functional properties of 158P3D2 and 158P3D2 mutations or analogs.

Recombinant human 158P3D2 protein or an analog or homolog or fragment thereof can be produced by mammalian cells transfected with a construct encoding a 158P3D2-related nucleotide. For example, 293T cells can be transfected with an expression plasmid encoding 158P3D2 or fragment, analog or homolog thereof, a 158P3D2-related protein is expressed in the 293T cells, and the recombinant 158P3D2 protein is isolated using standard purification methods (e.g., affinity purification using anti-158P3D2 antibodies). In another embodiment, a 158P3D2 coding sequence is subcloned into the retroviral vector pSRαMSVtkneo and used to infect various mammalian cell lines, such as NIH 3T3, TsuPr1, 293 and rat-1 in order to establish 158P3D2 expressing cell lines. Various other expression systems well known in the art can also be employed. Expression constructs encoding a leader peptide joined in frame to a 158P3D2 coding sequence can be used for the generation of a secreted form of recombinant 158P3D2 protein.

As discussed herein, redundancy in the genetic code permits variation in 158P3D2 gene sequences. In particular, it is known in the art that specific host species often have specific codon preferences, and thus one can adapt the disclosed sequence as preferred for a desired host. For example, preferred analog codon sequences typically have rare codons (i.e., codons having a usage frequency of less than about 20% in known sequences of the desired host) replaced with higher frequency codons. Codon preferences for a specific species are calculated, for example, by utilizing a codon usage table.

Additional sequence modifications are known to enhance protein expression in a cellular host. These include elimination of sequences encoding spurious polyadenylation signals, exon/intron splice site signals, transposon-like repeats, and/or other such well-characterized sequences that are deleterious to gene expression. The GC content of the sequence is adjusted to levels average for a given cellular host, as calculated by reference to known genes expressed in the host cell. Where possible, the sequence is modified to avoid predicted hairpin secondary mRNA structures. Other useful modifications include the addition of a translational initiation consensus sequence at the start of the open reading frame, as described in Kozak, Mol. Cell Biol., 9:5073-5080 (1989). Skilled artisans understand that the general rule that eukaryotic ribosomes initiate translation exclusively at the 5′ proximal AUG codon is abrogated only under rare conditions (see, e.g., Kozak PNAS 92(7): 2662-2666, (1995) and Kozak NAR 15(20): 8125-8148 (1987)).

III.) 158P3D2-RELATED PROTEINS

Another aspect of the present invention provides 158P3D2-related proteins. Specific embodiments of 158P3D2 proteins comprise a polypeptide having all or part of the amino acid sequence of human 158P3D2 as shown in FIG. 2 or FIG. 3. Alternatively, embodiments of 158P3D2 proteins comprise variant, homolog or analog polypeptides that have alterations in the amino acid sequence of 158P3D2 shown in FIG. 2 or FIG. 3.

In general, naturally occurring allelic variants of human 158P3D2 share a high degree of structural identity and homology (e.g., 90% or more homology). Typically, allelic variants of a 158P3D2 protein contain conservative amino acid substitutions within the 158P3D2 sequences described herein or contain a substitution of an amino acid from a corresponding position in a homologue of 158P3D2. One class of 158P3D2 allelic variants are proteins that share a high degree of homology with at least a small region of a particular 158P3D2 amino acid sequence, but further contain a radical departure from the sequence, such as a non-conservative substitution, truncation, insertion or frame shift. In comparisons of protein sequences, the terms, similarity, identity, and homology each have a distinct meaning as appreciated in the field of genetics. Moreover, orthology and paralogy can be important concepts describing the relationship of members of a given protein family in one organism to the members of the same family in other organisms.

Amino acid abbreviations are provided in Table II. Conservative amino acid substitutions can frequently be made in a protein without altering either the conformation or the function of the protein. Proteins of the invention can comprise 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15 conservative substitutions. Such changes include substituting any of isoleucine (1), valine (V), and leucine (L) for any other of these hydrophobic amino acids; aspartic acid (D) for glutamic acid (E) and vice versa; glutamine (Q) for asparagine (N) and vice versa; and serine (S) for threonine (T) and vice versa. Other substitutions can also be considered conservative, depending on the environment of the particular amino acid and its role in the three-dimensional structure of the protein. For example, glycine (G) and alanine (A) can frequently be interchangeable, as can alanine (A) and valine (V). Methionine (M), which is relatively hydrophobic, can frequently be interchanged with leucine and isoleucine, and sometimes with valine. Lysine (K) and arginine (R) are frequently interchangeable in locations in which the significant feature of the amino acid residue is its charge and the differing pK's of these two amino acid residues are not significant. Still other changes can be considered “conservative” in particular environments (see, e.g. Table III herein; pages 13-15 “Biochemistry” 2^(nd) ED. Lubert Stryered (Stanford University); Henikoff et al., PNAS 1992 Vol 89 10915-10919; Lei et al., J Biol Chem 1995 May 19; 270(20):11882-6).

Embodiments of the invention disclosed herein include a wide variety of art-accepted variants or analogs of 158P3D2 proteins such as polypeptides having amino acid insertions, deletions and substitutions. 158P3D2 variants can be made using methods known in the art such as site-directed mutagenesis, alanine scanning, and PCR mutagenesis. Site-directed mutagenesis (Carter et al., Nucl. Acids Res., 13:4331 (1986); Zoller et al., Nucl. Acids Res., 10:6487 (1987)), cassette mutagenesis (Wells et al., Gene, 34:315 (1985)), restriction selection mutagenesis (Wells et al., Philos. Trans. R. Soc. London SerA, 317:415 (1986)) or other known techniques can be performed on the cloned DNA to produce the 158P3D2 variant DNA.

Scanning amino acid analysis can also be employed to identify one or more amino acids along a contiguous sequence that is involved in a specific biological activity such as a protein-protein interaction. Among the preferred scanning amino acids are relatively small, neutral amino acids. Such amino acids include alanine, glycine, serine, and cysteine. Alanine is typically a preferred scanning amino acid among this group because it eliminates the side-chain beyond the beta-carbon and is less likely to alter the main-chain conformation of the variant. Alanine is also typically preferred because it is the most common amino acid. Further, it is frequently found in both buried and exposed positions (Creighton, The Proteins, (W.H. Freeman & Co., N.Y.); Chothia, J. Mol. Biol., 150:1 (1976)). If alanine substitution does not yield adequate amounts of variant, an isosteric amino acid can be used.

As defined herein, 158P3D2 variants, analogs or homologs, have the distinguishing attribute of having at least one epitope that is “cross reactive” with a 158P3D2 protein having an amino acid sequence of FIG. 3. As used in this sentence, “cross reactive” means that an antibody or T cell that specifically binds to an 158P3D2 variant also specifically binds to a 158P3D2 protein having an amino acid sequence set forth in FIG. 3. A polypeptide ceases to be a variant of a protein shown in FIG. 3, when it no longer contains any epitope capable of being recognized by an antibody or T cell that specifically binds to the starting 158P3D2 protein. Those skilled in the art understand that antibodies that recognize proteins bind to epitopes of varying size, and a grouping of the order of about four or five amino acids, contiguous or not, is regarded as a typical number of amino acids in a minimal epitope. See, e.g., Nair et al., J. Immunol 2000 165(12): 6949-6955; Hebbes et al., Mol Immunol (1989) 26(9):865-73; Schwartz et al., J Immunol (1985) 135(4):2598-608.

Other classes of 158P3D2-related protein variants share 70%, 75%, 80%, 85% or 90% or more similarity with an amino acid sequence of FIG. 3, or a fragment thereof. Another specific class of 158P3D2 protein variants or analogs comprise one or more of the 158P3D2 biological motifs described herein or presently known in the art. Thus, encompassed by the present invention are analogs of 158P3D2 fragments (nucleic or amino acid) that have altered functional (e.g. immunogenic) properties relative to the starting fragment. It is to be appreciated that motifs now or which become part of the art are to be applied to the nucleic or amino acid sequences of FIG. 2 or FIG. 3.

As discussed herein, embodiments of the claimed invention include polypeptides containing less than the full amino acid sequence of a 158P3D2 protein shown in FIG. 2 or FIG. 3. For example, representative embodiments of the invention comprise peptides/proteins having any 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15 or more contiguous amino acids of a 158P3D2 protein shown in FIG. 2 or FIG. 3.

Moreover, representative embodiments of the invention disclosed herein include polypeptides consisting of about amino acid 1 to about amino acid 10 of a 158P3D2 protein shown in FIG. 2 or FIG. 3, polypeptides consisting of about amino acid 10 to about amino acid 20 of a 158P3D2 protein shown in FIG. 2 or FIG. 3, polypeptides consisting of about amino acid 20 to about amino acid 30 of a 158P3D2 protein shown in FIG. 2 or FIG. 3, polypeptides consisting of about amino acid 30 to about amino acid 40 of a 158P3D2 protein shown in FIG. 2 or FIG. 3, polypeptides consisting of about amino acid 40 to about amino acid 50 of a 158P3D2 protein shown in FIG. 2 or FIG. 3, polypeptides consisting of about amino acid 50 to about amino acid 60 of a 158P3D2 protein shown in FIG. 2 or FIG. 3, polypeptides consisting of about amino acid 60 to about amino acid 70 of a 158P3D2 protein shown in FIG. 2 or FIG. 3, polypeptides consisting of about amino acid 70 to about amino acid 80 of a 158P3D2 protein shown in FIG. 2 or FIG. 3, polypeptides consisting of about amino acid 80 to about amino acid 90 of a 158P3D2 protein shown in FIG. 2 or FIG. 3, polypeptides consisting of about amino acid 90 to about amino acid 100 of a 158P3D2 protein shown in FIG. 2 or FIG. 3, etc. throughout the entirety of a 158P3D2 amino acid sequence. Moreover, polypeptides consisting of about amino acid 1 (or 20 or 30 or 40 etc.) to about amino acid 20, (or 130, or 140 or 150 etc.) of a 158P3D2 protein shown in FIG. 2 or FIG. 3 are embodiments of the invention. It is to be appreciated that the starting and stopping positions in this paragraph refer to the specified position as well as that position plus or minus 5 residues. 158P3D2-related proteins are generated using standard peptide synthesis technology or using chemical cleavage methods well known in the art. Alternatively, recombinant methods can be used to generate nucleic acid molecules that encode a 158P3D2-related protein. In one embodiment, nucleic acid molecules provide a means to generate defined fragments of a 158P3D2 protein (or variants, homologs or analogs thereof).

III.A.) Motif-bearing Protein Embodiments

Additional illustrative embodiments of the invention disclosed herein include 158P3D2 polypeptides comprising the amino acid residues of one or more of the biological motifs contained within a 158P3D2 polypeptide sequence set forth in FIG. 2 or FIG. 3. Various motifs are known in the art, and a protein can be evaluated for the presence of such motifs by a number of publicly available Internet sites (see, e.g., Epimatrix™ and Epimer™, Brown University, and BIMAS).

Motif bearing subsequences of all 158P3D2 variant proteins are set forth and identified in Table XVIII.

Table XX sets forth several frequently occurring motifs based on pfam searches (see URL address pfam.wustl.edu/). The columns of Table XX list (1) motif name abbreviation, (2) percent identity found amongst the different member of the motif family, (3) motif name or description and (4) most common function; location information is included if the motif is relevant for location.

Polypeptides comprising one or more of the 158P3D2 motifs discussed above are useful in elucidating the specific characteristics of a malignant phenotype in view of the observation that the 158P3D2 motifs discussed above are associated with growth dysregulation and because 158P3D2 is overexpressed in certain cancers (See, e.g., Table I). Casein kinase II, cAMP and camp-dependent protein kinase, and Protein Kinase C, for example, are enzymes known to be associated with the development of the malignant phenotype (see e.g. Chen et al., Lab Invest., 78(2): 165-174 (1998); Gaiddon et al., Endocrinology 136(10): 4331-4338 (1995); Hall et al., Nucleic Acids Research 24(6): 1119-1126 (1996); Peterziel et al., Oncogene 18(46): 6322-6329 (1999) and O'Brian, Oncol. Rep. 5(2): 305-309 (1998)). Moreover, both glycosylation and myristoylation are protein modifications also associated with cancer and cancer progression (see e.g. Dennis et al., Biochem. Biophys. Acta 1473(1):21-34 (1999); Raju et al., Exp. Cell Res. 235(1): 145-154 (1997)). Amidation is another protein modification also associated with cancer and cancer progression (see e.g., Treston et al., J. Natl. Cancer Inst. Monogr. (13): 169-175 (1992)).

In another embodiment, proteins of the invention comprise one or more of the immunoreactive epitopes identified in accordance with art-accepted methods, such as the peptides set forth in Tables V-XIX. CTL epitopes can be determined using specific algorithms to identify peptides within an 158P3D2 protein that are capable of optimally binding to specified HLA alleles (e.g., Table IV; Epimatrix™ and Epimer™, Brown University, and BIMAS). Moreover, processes for identifying peptides that have sufficient binding affinity for HLA molecules and which are correlated with being immunogenic epitopes, are well known in the art, and are carried out without undue experimentation. In addition, processes for identifying peptides that are immunogenic epitopes, are well known in the art, and are carried out without undue experimentation either in vitro or in vivo.

Also known in the art are principles for creating analogs of such epitopes in order to modulate immunogenicity. For example, one begins with an epitope that bears a CTL or HTL motif (see, e.g., the HLA Class I and HLA Class II motifs/supermotifs of Table IV). The epitope is analoged by substituting out an amino acid at one of the specified positions, and replacing it with another amino acid specified for that position. For example, one can substitute out a deleterious residue in favor of any other residue, such as a preferred residue as defined in Table IV; substitute a less-preferred residue with a preferred residue as defined in Table IV; or substitute an originally-occurring preferred residue with another preferred residue as defined in Table IV. Substitutions can occur at primary anchor positions or at other positions in a peptide; see, e.g., Table IV.

A variety of references reflect the art regarding the identification and generation of epitopes in a protein of interest as well as analogs thereof. See, for example, WO 9733602 to Chesnut et al.; Sette, Immunogenetics 1999 50(3-4): 201-212; Sette et al., J. Immunol. 2001 166(2): 1389-1397; Sidney et al., Hum. Immunol. 1997 58(1): 12-20; Kondo et al., Immunogenetics 1997 45(4): 249-258; Sidney et al., J. Immunol. 1996 157(8): 3480-90; and Falk et al., Nature 351: 290-6 (1991); Hunt et al., Science 255:1261-3 (1992); Parker et al., J. Immunol. 149:3580-7 (1992); Parker et al., J. Immunol. 152:163-75 (1994)); Kast et al., 1994 152(8): 3904-12; Borras-Cuesta et al., Hum. Immunol. 2000 61(3): 266-278; Alexander et al., J. Immunol. 2000 164(3); 164(3): 1625-1633; Alexander et al., PMID: 7895164, UI: 95202582; O'Sullivan et al., J. Immunol. 1991 147(8): 2663-2669; Alexander et al., Immunity 1994 1(9): 751-761 and Alexander et al., Immunol. Res. 1998 18(2): 79-92.

Related embodiments of the inventions include polypeptides comprising combinations of the different motifs set forth in Table XXI, and/or, one or more of the predicted CTL epitopes of Table V through Table XIX, and/or, one or more of the T cell binding motifs known in the art. Preferred embodiments contain no insertions, deletions or substitutions either within the motifs or the intervening sequences of the polypeptides. In addition, embodiments which include a number of either N-terminal and/or C-terminal amino acid residues on either side of these motifs may be desirable (to, for example, include a greater portion of the polypeptide architecture in which the motif is located). Typically the number of N-terminal and/or C-terminal amino acid residues on either side of a motif is between about 1 to about 100 amino acid residues, preferably 5 to about 50 amino acid residues.

158P3D2-related proteins are embodied in many forms, preferably in isolated form. A purified 158P3D2 protein molecule will be substantially free of other proteins or molecules that impair the binding of 158P3D2 to antibody, T cell or other ligand. The nature and degree of isolation and purification will depend on the intended use. Embodiments of a 158P3D2-related proteins include purified 158P3D2-related proteins and functional, soluble 158P3D2-related proteins. In one embodiment, a functional, soluble 158P3D2 protein or fragment thereof retains the ability to be bound by antibody, T cell or other ligand.

The invention also provides 158P3D2 proteins comprising biologically active fragments of a 158P3D2 amino acid sequence shown in FIG. 2 or FIG. 3. Such proteins exhibit properties of the starting 158P3D2 protein, such as the ability to elicit the generation of antibodies that specifically bind an epitope associated with the starting 158P3D2 protein; to be bound by such antibodies; to elicit the activation of HTL or CTL; and/or, to be recognized by HTL or CTL that also specifically bind to the starting protein.

158P3D2-related polypeptides that contain particularly interesting structures can be predicted and/or identified using various analytical techniques well known in the art, including, for example, the methods of Chou-Fasman, Garnier-Robson, Kyte-Doolittle, Eisenberg, Karplus-Schultz or Jameson-Wolf analysis, or on the basis of immunogenicity. Fragments that contain such structures are particularly useful in generating subunit-specific anti-158P3D2 antibodies, or T cells or in identifying cellular factors that bind to 158P3D2. For example, hydrophilicity profiles can be generated, and immunogenic peptide fragments identified, using the method of Hopp, T. P. and Woods, K. R., 1981, Proc. Natl. Acad. Sci. U.S.A. 78:3824-3828. Hydropathicity profiles can be generated, and immunogenic peptide fragments identified, using the method of Kyte, J. and Doolittle, R. F., 1982, J. Mol. Biol. 157:105-132. Percent (%) Accessible Residues profiles can be generated, and immunogenic peptide fragments identified, using the method of Janin J., 1979, Nature 277:491-492. Average Flexibility profiles can be generated, and immunogenic peptide fragments identified, using the method of Bhaskaran R., Ponnuswamy P. K., 1988, Int. J. Pept. Protein Res. 32:242-255. Beta-turn profiles can be generated, and immunogenic peptide fragments identified, using the method of Deleage, G., Roux B., 1987, Protein Engineering 1:289-294.

CTL epitopes can be determined using specific algorithms to identify peptides within an 158P3D2 protein that are capable of optimally binding to specified HLA alleles (e.g., by using the SYFPEITHI site at World Wide Web; the listings in Table IV(A)-(E); Epimatrix™ and Epimer™, Brown University, and BIMAS). Illustrating this, peptide epitopes from 158P3D2 that are presented in the context of human MHC class I molecules HLA-A1, A2, A3, A11, A24, B7 and B35 were predicted (Tables V-XIX). Specifically, the complete amino acid sequence of the 158P3D2 protein and relevant portions of other variants, i.e., for HLA Class I predictions 9 flanking residues on either side of a point mutation, and for HLA Class II predictions 14 flanking residues on either side of a point mutation, were entered into the HLA Peptide Motif Search algorithm found in the Bioinformatics and Molecular Analysis Section (BIMAS) web site; for HLA Class II the site SYFPEITHI.

The HLA peptide motif search algorithm was developed by Dr. Ken Parker based on binding of specific peptide sequences in the groove of HLA Class I molecules, in particular HLA-A2 (see, e.g., Falk et al., Nature 351: 290-6 (1991); Hunt et al., Science 255:1261-3 (1992); Parker et al., J. Immunol. 149:3580-7 (1992); Parker et al., J. Immunol. 152:163-75 (1994)). This algorithm allows location and ranking of 8-mer, 9-mer, and 10 mer peptides from a complete protein sequence for predicted binding to HLA-A2 as well as numerous other HLA Class I molecules. Many HLA class I binding peptides are 8-, 9-, 10 or 11-mers. For example, for class I HLA-A2, the epitopes preferably contain a leucine (L) or methionine (M) at position 2 and a valine (V) or leucine (L) at the C-terminus (see, e.g., Parker et al., J. Immunol. 149:3580-7 (1992)). Selected results of 158P3D2 predicted binding peptides are shown in Tables V-XIX herein. In Tables V-XIX, the top ranking candidates, 9-mers, 10-mers and 15-mers (for each family member), are shown along with their location, the amino acid sequence of each specific peptide, and an estimated binding score. The binding score corresponds to the estimated half time of dissociation of complexes containing the peptide at 37° C. at pH 6.5. Peptides with the highest binding score are predicted to be the most tightly bound to HLA Class I on the cell surface for the greatest period of time and thus represent the best immunogenic targets for T-cell recognition.

Actual binding of peptides to an HLA allele can be evaluated by stabilization of HLA expression on the antigen-processing defective cell line T2 (see, e.g., Xue et al., Prostate 30:73-8 (1997) and Peshwa et al., Prostate 36:129-38 (1998)). Immunogenicity of specific peptides can be evaluated in vitro by stimulation of CD8+ cytotoxic T lymphocytes (CTL) in the presence of antigen presenting cells such as dendritic cells.

It is to be appreciated that every epitope predicted by the BIMAS site, Epimer™ and Epimatrix™ sites, or specified by the HLA class I or class II motifs available in the art or which become part of the art such as set forth in Table IV (or determined using World Wide Web site URL syfpeithi.bmi-heidelberg.com/, or BIMAS, bimas.dcrt.nih.gov/) are to be “applied” to a 158P3D2 protein in accordance with the invention. As used in this context “applied” means that a 158P3D2 protein is evaluated, e.g., visually or by computer-based patterns finding methods, as appreciated by those of skill in the relevant art. Every subsequence of a 158P3D2 protein of 8, 9, 10, or II amino acid residues that bears an HLA Class I motif, or a subsequence of 9 or more amino acid residues that bear an HLA Class II motif are within the scope of the invention.

III.B.) Expression of 158P3D2-Related Proteins

In an embodiment described in the examples that follow, 158P3D2 can be conveniently expressed in cells (such as 293T cells) transfected with a commercially available expression vector such as a CMV-driven expression vector encoding 158P3D2 with a C-terminal 6×His and MYC tag (pcDNA3.1/mycHIS, Invitrogen or Tag5, GenHunter Corporation, Nashville Tenn.). The Tag5 vector provides an IgGK secretion signal that can be used to facilitate the production of a secreted 158P3D2 protein in transfected cells. The secreted HIS-tagged 158P3D2 in the culture media can be purified, e.g., using a nickel column using standard techniques.

III.C.) Modifications of 158P3D2-Related Proteins

Modifications of 158P3D2-related proteins such as covalent modifications are included within the scope of this invention. One type of covalent modification includes reacting targeted amino acid residues of a 158P3D2 polypeptide with an organic derivatizing agent that is capable of reacting with selected side chains or the N- or C-terminal residues of a 158P3D2 protein. Another type of covalent modification of a 158P3D2 polypeptide included within the scope of this invention comprises altering the native glycosylation pattern of a protein of the invention. Another type of covalent modification of 158P3D2 comprises linking a 158P3D2 polypeptide to one of a variety of nonproteinaceous polymers, e.g., polyethylene glycol (PEG), polypropylene glycol, or polyoxyalkylenes, in the manner set forth in U.S. Pat. No. 4,640,835; 4,496,689; 4,301,144; 4,670,417; 4,791,192 or 4,179,337.

The 158P3D2-related proteins of the present invention can also be modified to form a chimeric molecule comprising 158P3D2 fused to another, heterologous polypeptide or amino acid sequence. Such a chimeric molecule can be synthesized chemically or recombinantly. A chimeric molecule can have a protein of the invention fused to another tumor-associated antigen or fragment thereof. Alternatively, a protein in accordance with the invention can comprise a fusion of fragments of a 158P3D2 sequence (amino or nucleic acid) such that a molecule is created that is not, through its length, directly homologous to the amino or nucleic acid sequences shown in FIG. 2 or FIG. 3. Such a chimeric molecule can comprise multiples of the same subsequence of 158P3D2. A chimeric molecule can comprise a fusion of a 158P3D2-related protein with a polyhistidine epitope tag, which provides an epitope to which immobilized nickel can selectively bind, with cytokines or with growth factors. The epitope tag is generally placed at the amino- or carboxyl-terminus of a 158P3D2 protein. In an alternative embodiment, the chimeric molecule can comprise a fusion of a 158P3D2-related protein with an immunoglobulin or a particular region of an immunoglobulin. For a bivalent form of the chimeric molecule (also referred to as an “immunoadhesin”), such a fusion could be to the Fc region of an IgG molecule. The Ig fusions preferably include the substitution of a soluble (transmembrane domain deleted or inactivated) form of a 158P3D2 polypeptide in place of at least one variable region within an Ig molecule. In a preferred embodiment, the immunoglobulin fusion includes the hinge, CH2 and CH3, or the hinge, CH1, CH2 and CH3 regions of an IgGI molecule. For the production of immunoglobulin fusions see, e.g., U.S. Pat. No. 5,428,130 issued Jun. 27, 1995.

III.D.) Uses of 158P3D2-Related Proteins

The proteins of the invention have a number of different specific uses. As 158P3D2 is highly expressed in prostate and other cancers, 158P3D2-related proteins are used in methods that assess the status of 158P3D2 gene products in normal versus cancerous tissues, thereby elucidating the malignant phenotype. Typically, polypeptides from specific regions of a 158P3D2 protein are used to assess the presence of perturbations (such as deletions, insertions, point mutations etc.) in those regions (such as regions containing one or more motifs). Exemplary assays utilize antibodies or T cells targeting 158P3D2-related proteins comprising the amino acid residues of one or more of the biological motifs contained within a 158P3D2 polypeptide sequence in order to evaluate the characteristics of this region in normal versus cancerous tissues or to elicit an immune response to the epitope. Alternatively, 158P3D2-related proteins that contain the amino acid residues of one or more of the biological motifs in a 158P3D2 protein are used to screen for factors that interact with that region of 158P3D2.

158P3D2 protein fragments/subsequences are particularly useful in generating and characterizing domain-specific antibodies (e.g., antibodies recognizing an extracellular or intracellular epitope of an 158P3D2 protein), for identifying agents or cellular factors that bind to 158P3D2 or a particular structural domain thereof, and in various therapeutic and diagnostic contexts, including but not limited to diagnostic assays, cancer vaccines and methods of preparing such vaccines.

Proteins encoded by the 158P3D2 genes, or by analogs, homologs or fragments thereof, have a variety of uses, including but not limited to generating antibodies and in methods for identifying ligands and other agents and cellular constituents that bind to an 158P3D2 gene product. Antibodies raised against an 158P3D2 protein or fragment thereof are useful in diagnostic and prognostic assays, and imaging methodologies in the management of human cancers characterized by expression of 158P3D2 protein, such as those listed in Table I. Such antibodies can be expressed intracellularly and used in methods of treating patients with such cancers. 158P3D2-related nucleic acids or proteins are also used in generating HTL or CTL responses.

Various immunological assays useful for the detection of 158P3D2 proteins are used, including but not limited to various types of radioimmunoassays, enzyme-linked immunosorbent assays (ELISA), enzyme-linked immunofluorescent assays (ELIFA), immunocytochemical methods, and the like. Antibodies can be labeled and used as immunological imaging reagents capable of detecting 158P3D2-expressing cells (e.g., in radioscintigraphic imaging methods). 158P3D2 proteins are also particularly useful in generating cancer vaccines, as further described herein.

IV.) 158P3D2 ANTIBODIES

Another aspect of the invention provides antibodies that bind to 158P3D2-related proteins. Preferred antibodies specifically bind to a 158P3D2-related protein and do not bind (or bind weakly) to peptides or proteins that are not 158P3D2-related proteins. For example, antibodies that bind 158P3D2 can bind 158P3D2-related proteins such as the homologs or analogs thereof.

158P3D2 antibodies of the invention are particularly useful in cancer (see, e.g., Table I) diagnostic and prognostic assays, and imaging methodologies. Similarly, such antibodies are useful in the treatment, diagnosis, and/or prognosis of other cancers, to the extent 158P3D2 is also expressed or overexpressed in these other cancers. Moreover, intracellularly expressed antibodies (e.g., single chain antibodies) are therapeutically useful in treating cancers in which the expression of 158P3D2 is involved, such as advance or metastatic prostate cancers.

The invention also provides various immunological assays useful for the detection and quantification of 158P3D2 and mutant 158P3D2-related proteins. Such assays can comprise one or more 158P3D2 antibodies capable of recognizing and binding a 158P3D2-related protein, as appropriate. These assays are performed within various immunological assay formats well known in the art, including but not limited to various types of radioimmunoassays, enzyme-linked immunosorbent assays (ELISA), enzyme-linked immunofluorescent assays (ELIFA), and the like.

Immunological non-antibody assays of the invention also comprise T cell immunogenicity assays (inhibitory or stimulatory) as well as major histocompatibility complex (MHC) binding assays.

In addition, immunological imaging methods capable of detecting prostate cancer and other cancers expressing 158P3D2 are also provided by the invention, including but not limited to radioscintigraphic imaging methods using labeled 158P3D2 antibodies. Such assays are clinically useful in the detection, monitoring, and prognosis of 158P3D2 expressing cancers such as prostate cancer.

158P3D2 antibodies are also used in methods for purifying a 158P3D2-related protein and for isolating 158P3D2 homologues and related molecules. For example, a method of purifying a 158P3D2-related protein comprises incubating an 158P3D2 antibody, which has been coupled to a solid matrix, with a lysate or other solution containing a 158P3D2-related protein under conditions that permit the 158P3D2 antibody to bind to the 158P3D2-related protein; washing the solid matrix to eliminate impurities; and eluting the 158P3D2-related protein from the coupled antibody. Other uses of 158P3D2 antibodies in accordance with the invention include generating anti-idiotypic antibodies that mimic a 158P3D2 protein.

Various methods for the preparation of antibodies are well known in the art. For example, antibodies can be prepared by immunizing a suitable mammalian host using a 158P3D2-related protein, peptide, or fragment, in isolated or immunoconjugated form (Antibodies: A Laboratory Manual, CSH Press, Eds., Harlow, and Lane (1988); Harlow, Antibodies, Cold Spring Harbor Press, NY (1989)). In addition, fusion proteins of 158P3D2 can also be used, such as a 158P3D2 GST-fusion protein. In a particular embodiment, a GST fusion protein comprising all or most of the amino acid sequence of FIG. 2 or FIG. 3 is produced, then used as an immunogen to generate appropriate antibodies. In another embodiment, a 158P3D2-related protein is synthesized and used as an immunogen.

In addition, naked DNA immunization techniques known in the art are used (with or without purified 158P3D2-related protein or 158P3D2 expressing cells) to generate an immune response to the encoded immunogen (for review, see Donnelly et al., 1997, Ann. Rev. Immunol. 15: 617-648).

The amino acid sequence of a 158P3D2 protein as shown in FIG. 2 or FIG. 3 can be analyzed to select specific regions of the 158P3D2 protein for generating antibodies. For example, hydrophobicity and hydrophilicity analyses of a 158P3D2 amino acid sequence are used to identify hydrophilic regions in the 158P3D2 structure. Regions of a 158P3D2 protein that show immunogenic structure, as well as other regions and domains, can readily be identified using various other methods known in the art, such as Chou-Fasman, Garnier-Robson, Kyte-Doolittle, Eisenberg, Karplus-Schultz or Jameson-Wolf analysis. Hydrophilicity profiles can be generated using the method of Hopp, T. P. and Woods, K. R., 1981, Proc. Natl. Acad. Sci. U.S.A. 78:3824-3828. Hydropathicity profiles can be generated using the method of Kyte, J. and Doolittle, R. F., 1982, J. Mol. Biol. 157:105-132. Percent (%) Accessible Residues profiles can be generated using the method of Janin J., 1979, Nature 277:491-492. Average Flexibility profiles can be generated using the method of Bhaskaran R., Ponnuswamy P. K., 1988, Int. J. Pept. Protein Res. 32:242-255. Beta-turn profiles can be generated using the method of Deleage, G., Roux B., 1987, Protein Engineering 1:289-294. Thus, each region identified by any of these programs or methods is within the scope of the present invention. Methods for the generation of 158P3D2 antibodies are further illustrated by way of the examples provided herein. Methods for preparing a protein or polypeptide for use as an immunogen are well known in the art. Also well known in the art are methods for preparing immunogenic conjugates of a protein with a carrier, such as BSA, KLH or other carrier protein. In some circumstances, direct conjugation using, for example, carbodiimide reagents are used; in other instances linking reagents such as those supplied by Pierce Chemical Co., Rockford, Ill., are effective. Administration of a 158P3D2 immunogen is often conducted by injection over a suitable time period and with use of a suitable adjuvant, as is understood in the art. During the immunization schedule, titers of antibodies can be taken to determine adequacy of antibody formation.

158P3D2 monoclonal antibodies can be produced by various means well known in the art. For example, immortalized cell lines that secrete a desired monoclonal antibody are prepared using the standard hybridoma technology of Kohler and Milstein or modifications that immortalize antibody-producing B cells, as is generally known. Immortalized cell lines that secrete the desired antibodies are screened by immunoassay in which the antigen is a 158P3D2-related protein. When the appropriate immortalized cell culture is identified, the cells can be expanded and antibodies produced either from in vitro cultures or from ascites fluid.

The antibodies or fragments of the invention can also be produced, by recombinant means. Regions that bind specifically to the desired regions of a 158P3D2 protein can also be produced in the context of chimeric or complementarity determining region (CDR) grafted antibodies of multiple species origin. Humanized or human 158P3D2 antibodies can also be produced, and are preferred for use in therapeutic contexts. Methods for humanizing murine and other non-human antibodies, by substituting one or more of the non-human antibody CDRs for corresponding human antibody sequences, are well known (see for example, Jones et al., 1986, Nature 321: 522-525; Riechmann et al., 1988, Nature 332: 323-327; Verhoeyen et al., 1988, Science 239: 1534-1536). See also, Carter et al., 1993, Proc. Natl. Acad. Sci. USA 89: 4285 and Sims et al., 1993, J. Immunol. 151: 2296.

Methods for producing fully human monoclonal antibodies include phage display and transgenic methods (for review, see Vaughan et al., 1998, Nature Biotechnology 16: 535-539). Fully human 158P3D2 monoclonal antibodies can be generated using cloning technologies employing large human Ig gene combinatorial libraries (i.e., phage display) (Griffiths and Hoogenboom, Building an in vitro immune system: human antibodies from phage display libraries. In: Protein Engineering of Antibody Molecules for Prophylactic and Therapeutic Applications in Man, Clark, M. (Ed.), Nottingham Academic, pp 45-64 (1993); Burton and Barbas, Human Antibodies from combinatorial libraries. Id., pp 65-82). Fully human 158P3D2 monoclonal antibodies can also be produced using transgenic mice engineered to contain human immunoglobulin gene loci as described in PCT Patent Application WO 98/24893, Kucherlapati and Jakobovits et al., published Dec. 3, 1997 (see also, Jakobovits, 1998, Exp. Opin. Invest. Drugs 7(4): 607-614; U.S. Pat. Nos. 6,162,963 issued 19 Dec. 2000; 6,150,584 issued 12 Nov. 2000; and, 6,114598 issued 5 Sep. 2000). This method avoids the in vitro manipulation required with phage display technology and efficiently produces high affinity authentic human antibodies.

Reactivity of 158P3D2 antibodies with an 158P3D2-related protein can be established by a number of well known means, including Western blot, immunoprecipitation, ELISA, and FACS analyses using, as appropriate, 158P3D2-related proteins, 158P3D2-expressing cells or extracts thereof. A 158P3D2 antibody or fragment thereof can be labeled with a detectable marker or conjugated to a second molecule. Suitable detectable markers include, but are not limited to, a radioisotope, a fluorescent compound, a bioluminescent compound, chemiluminescent compound, a metal chelator or an enynie. Further, bi-specific antibodies specific for two or more 158P3D2 epitopes are generated using methods generally known in the art. Homodimeric antibodies can also be generated by cross-linking techniques known in the art (e.g., Wolff et al., Cancer Res. 53: 2560-2565).

V.) 158P3D2 CELLULAR IMMUNE RESPONSES

The mechanism by which T cells recognize antigens has been delineated. Efficacious peptide epitope vaccine compositions of the invention induce a therapeutic or prophylactic immune responses in very broad segments of the world-wide population. For an understanding of the value and efficacy of compositions of the invention that induce cellular immune responses, a brief review of immunology-related technology is provided.

A complex of an HLA molecule and a peptidic antigen acts as the ligand recognized by HLA-restricted T cells (Buus, S. et al., Cell 47:1071, 1986; Babbitt, B. P. et al., Nature 317:359, 1985; Townsend, A. and Bodmer, H., Annu. Rev. Immunol. 7:601, 1989; Germain, R. N., Annu. Rev. Immunol. 11:403, 1993). Through the study of single amino acid substituted antigen analogs and the sequencing of endogenously bound, naturally processed peptides, critical residues that correspond to motifs required for specific binding to HLA antigen molecules have been identified and are set forth in Table IV (see also, e.g., Southwood, et al., J. Immunol. 160:3363, 1998; Rammensee, et al., Immunogenetics 41:178, 1995; Rammensee et al., SYFPEITHI, access via World Wide Web at URL syfpeithi.bmi-heidelberg.com/; Sette, A. and Sidney, J. Curr. Opin. Immunol. 10:478, 1998; Engelhard, V. H., Curr. Opin. Immunol. 6:13, 1994; Sette, A. and Grey, H. M., Curr. Opin. Immunol. 4:79, 1992; Sinigaglia, F. and Hammer, J. Curr. Biol. 6:52, 1994; Ruppert et al., Cell 74:929-937, 1993; Kondo et al., J. Immunol. 155:4307-4312, 1995; Sidney et al., J. Immunol. 157:3480-3490, 1996; Sidney et al., Human Immunol. 45:79-93, 1996; Sette, A. and Sidney, J. Immunogenetics 1999 November; 50(3-4):201-12, Review).

Furthermore, x-ray crystallographic analyses of HLA-peptide complexes have revealed pockets within the peptide binding cleft/groove of HLA molecules which accommodate, in an allele-specific mode, residues borne by peptide ligands; these residues in turn determine the HLA binding capacity of the peptides in which they are present. (See, e.g., Madden, D. R. Annu. Rev. Immunol. 13:587, 1995; Smith, et al., Immunity 4:203, 1996; Fremont et al., Immunity 8:305, 1998; Stern et al., Structure 2:245, 1994; Jones, E. Y. Curr. Opin. Immunol. 9:75, 1997; Brown, J. H. et al., Nature 364:33, 1993; Guo, H. C. et al., Proc. Natl. Acad. Sci. USA 90:8053, 1993; Guo, H. C. et al., Nature 360:364, 1992; Silver, M. L. et al., Nature 360:367, 1992; Matsumura, M. et al., Science 257:927, 1992; Madden et al., Cell 70:1035, 1992; Fremont, D. H. et al., Science 257:919, 1992; Saper, M. A., Bjorkman, P. J. and Wiley, D. C., J. Mol. Biol. 219:277, 1991.)

Accordingly, the definition of class I and class II allele-specific HLA binding motifs, or class I or class II supermotifs allows identification of regions within a protein that are correlated with binding to particular HLA antigen(s).

Thus, by a process of HLA motif identification, candidates for epitope-based vaccines have been identified; such candidates can be further evaluated by HLA-peptide binding assays to determine binding affinity and/or the time period of association of the epitope and its corresponding HLA molecule. Additional confirmatory work can be performed to select, amongst these vaccine candidates, epitopes with preferred characteristics in terms of population coverage, and/or immunogenicity.

Various strategies can be utilized to evaluate cellular immunogenicity, including:

1) Evaluation of primary T cell cultures from normal individuals (see, e.g., Wentworth, P. A. et al., Mol. Immunol. 32:603, 1995; Celis, E. et al., Proc. Natl. Acad. Sci. USA 91:2105, 1994; Tsai, V. et al., J. Immunol. 158:1796, 1997; Kawashima, I. et al., Human Immunol. 59:1, 1998). This procedure involves the stimulation of peripheral blood lymphocytes (PBL) from normal subjects with a test peptide in the presence of antigen presenting cells in vitro over a period of several weeks. T cells specific for the peptide become activated during this time and are detected using, e.g., a lymphokine- or ⁵¹Cr-release assay involving peptide sensitized target cells.

2) Immunization of HLA transgenic mice (see, e.g., Wentworth, P. A. et al., J. Immunol. 26:97, 1996; Wentworth, P. A. et al., Int. Immunol. 8:651, 1996; Alexander, J. et al., J. Immunol. 159:4753, 1997). For example, in such methods peptides in incomplete Freund's adjuvant are administered subcutaneously to HLA transgenic mice. Several weeks following immunization, splenocytes are removed and cultured in vitro in the presence of test peptide for approximately one week. Peptide-specific T cells are detected using, e.g., a ⁵¹ Cr-release assay involving peptide sensitized target cells and target cells expressing endogenously generated antigen.

3) Demonstration of recall T cell responses from immune individuals who have been either effectively vaccinated and/or from chronically ill patients (see, e.g., Rehermann, B. et al., J. Exp. Med. 181:1047, 1995; Doolan, D. L. et al., Immunity 7:97, 1997; Bertoni, R. et al., J. Clin. Invest. 100:503, 1997; Threlkeld, S. C. et al., J. Immunol. 159:1648, 1997; Diepolder, H. M. et al., J. Virol. 71:6011, 1997). Accordingly, recall responses are detected by culturing PBL from subjects that have been exposed to the antigen due to disease and thus have generated an immune response “naturally”, or from patients who were vaccinated against the antigen. PBL from subjects are cultured in vitro for 1-2 weeks in the presence of test peptide plus antigen presenting cells (APC) to allow activation of “memory” T cells, as compared to “naive” T cells. At the end of the culture period, T cell activity is detected using assays including ⁵¹Cr release involving peptide-sensitized targets, T cell proliferation, or lymphokine release.

VI.) 158P3D2 TRANSGENIC ANIMALS

Nucleic acids that encode a 158P3D2-related protein can also be used to generate either transgenic animals or “knock out” animals that, in turn, are useful in the development and screening of therapeutically useful reagents. In accordance with established techniques, cDNA encoding 158P3D2 can be used to clone genomic DNA that encodes 158P3D2. The cloned genomic sequences can then be used to generate transgenic animals containing cells that express DNA that encode 158P3D2. Methods for generating transgenic animals, particularly animals such as mice or rats, have become conventional in the art and are described, for example, in U.S. Pat. Nos. 4,736,866 issued 12 Apr. 1988, and 4,870,009 issued 26 Sep. 1989. Typically, particular cells would be targeted for 158P3D2 transgene incorporation with tissue-specific enhancers.

Transgenic animals that include a copy of a transgene encoding 158P3D2 can be used to examine the effect of increased expression of DNA that encodes 158P3D2. Such animals can be used as tester animals for reagents thought to confer protection from, for example, pathological conditions associated with its overexpression. In accordance with this aspect of the invention, an animal is treated with a reagent and a reduced incidence of a pathological condition, compared to untreated animals that bear the transgene, would indicate a potential therapeutic intervention for the pathological condition.

Alternatively, non-human homologues of 158P3D2 can be used to construct a 158P3D2 “knock out” animal that has a defective or altered gene encoding 158P3D2 as a result of homologous recombination between the endogenous gene encoding 158P3D2 and altered genomic DNA encoding 158P3D2 introduced into an embryonic cell of the animal. For example, cDNA that encodes 158P3D2 can be used to clone genomic DNA encoding 158P3D2 in accordance with established techniques. A portion of the genomic DNA encoding 158P3D2 can be deleted or replaced with another gene, such as a gene encoding a selectable marker that can be used to monitor integration. Typically, several kilobases of unaltered flanking DNA (both at the 5′ and 3′ ends) are included in the vector (see, e.g., Thomas and Capecchi, Cell, 51:503 (1987) for a description of homologous recombination vectors). The vector is introduced into an embryonic stem cell line (e.g., by electroporation) and cells in which the introduced DNA has homologously recombined with the endogenous DNA are selected (see, e.g., Li et al., Cell 69:915 (1992)). The selected cells are then injected into a blastocyst of an animal (e.g., a mouse or rat) to form aggregation chimeras (see, e.g., Bradley, in Teratocarcinomas and Embryonic Stem Cells: A Practical Approach, E. J. Robertson, ed. (IRL, Oxford, 1987), pp. 113-152). A chimeric embryo can then be implanted into a suitable pseudopregnant female foster animal, and the embryo brought to term to create a “knock out” animal. Progeny harboring the homologously recombined DNA in their germ cells can be identified by standard techniques and used to breed animals in which all cells of the animal contain the homologously recombined DNA. Knock out animals can be characterized, for example, for their ability to defend against certain pathological conditions or for their development of pathological conditions due to absence of a 158P3D2 polypeptide.

VII.) METHODS FOR THE DETECTION OF 158P3D2

Another aspect of the present invention relates to methods for detecting 158P3D2 polynucleotides and 158P3D2-related proteins, as well as methods for identifying a cell that expresses 158P3D2. The expression profile of 158P3D2 makes it a diagnostic marker for metastasized disease. Accordingly, the status of 158P3D2 gene products provides information useful for predicting a variety of factors including susceptibility to advanced stage disease, rate of progression, and/or tumor aggressiveness. As discussed in detail herein, the status of 158P3D2 gene products in patient samples can be analyzed by a variety protocols that are well known in the art including immunohistochemical analysis, the variety of Northern blotting techniques including in situ hybridization, RT-PCR analysis (for example on laser capture micro-dissected samples), Western blot analysis and tissue array analysis.

More particularly, the invention provides assays for the detection of 158P3D2 polynucleotides in a biological sample, such as serum, bone, prostate, and other tissues, urine, semen, cell preparations, and the like. Detectable 158P3D2 polynucleotides include, for example, a 158P3D2 gene or fragment thereof, 158P3D2 mRNA, alternative splice variant 158P3D2 mRNAs, and recombinant DNA or RNA molecules that contain a 158P3D2 polynucleotide. A number of methods for amplifying and/or detecting the presence of 158P3D2 polynucleotides are well known in the art and can be employed in the practice of this aspect of the invention.

In one embodiment, a method for detecting an 158P3D2 mRNA in a biological sample comprises producing cDNA from the sample by reverse transcription using at least one primer; amplifying the cDNA so produced using an 158P3D2 polynucleotides as sense and antisense primers to amplify 158P3D2 cDNAs therein; and detecting the presence of the amplified 158P3D2 cDNA. Optionally, the sequence of the amplified 158P3D2 cDNA can be determined.

In another embodiment, a method of detecting a 158P3D2 gene in a biological sample comprises first isolating genomic DNA from the sample; amplifying the isolated genomic DNA using 158P3D2 polynucleotides as sense and antisense primers; and detecting the presence of the amplified 158P3D2 gene. Any number of appropriate sense and antisense probe combinations can be designed from a 158P3D2 nucleotide sequence (see, e.g., FIG. 2) and used for this purpose.

The invention also provides assays for detecting the presence of an 158P3D2 protein in a tissue or other biological sample such as serum, semen, bone, prostate, urine, cell preparations, and the like. Methods for detecting a 158P3D2-related protein are also well known and include, for example, immunoprecipitation, immunohistochemical analysis, Western blot analysis, molecular binding assays, ELISA, ELIFA and the like. For example, a method of detecting the presence of a 158P3D2-related protein in a biological sample comprises first contacting the sample with a 158P3D2 antibody, a 158P3D2-reactive fragment thereof, or a recombinant protein containing an antigen binding region of a 158P3D2 antibody; and then detecting the binding of 158P3D2-related protein in the sample.

Methods for identifying a cell that expresses 158P3D2 are also within the scope of the invention. In one embodiment, an assay for identifying a cell that expresses a 158P3D2 gene comprises detecting the presence of 158P3D2 mRNA in the cell. Methods for the detection of particular mRNAs in cells are well known and include, for example, hybridization assays using complementary DNA probes (such as in situ hybridization using labeled 158P3D2 riboprobes, Northern blot and related techniques) and various nucleic acid amplification assays (such as RT-PCR using complementary primers specific for 158P3D2, and other amplification type detection methods, such as, for example, branched DNA, SISBA, TMA and the like). Alternatively, an assay for identifying a cell that expresses a 158P3D2 gene comprises detecting the presence of 158P3D2-related protein in the cell or secreted by the cell. Various methods for the detection of proteins are well known in the art and are employed for the detection of 158P3D2-related proteins and cells that express 158P3D2-related proteins. 158P3D2 expression analysis is also useful as a tool for identifying and evaluating agents that modulate 158P3D2 gene expression. For example, 158P3D2 expression is significantly upregulated in prostate cancer, and is expressed in cancers of the tissues listed in Table I. Identification of a molecule or biological agent that inhibits 158P3D2 expression or over-expression in cancer cells is of therapeutic value. For example, such an agent can be identified by using a screen that quantifies 158P3D2 expression by RT-PCR, nucleic acid hybridization or antibody binding.

VIII.) METHODS FOR MONITORING THE STATUS OF 158P3D2-RELATED GENES AND THEIR PRODUCTS

Oncogenesis is known to be a multistep process where cellular growth becomes progressively dysregulated and cells progress from a normal physiological state to precancerous and then cancerous states (see, e.g., Alers et al., Lab Invest. 77(5): 437-438 (1997) and Isaacs et al., Cancer Surv. 23: 19-32 (1995)). In this context, examining a biological sample for evidence of dysregulated cell growth (such as aberrant 158P3D2 expression in cancers) allows for early detection of such aberrant physiology, before a pathologic state such as cancer has progressed to a stage that therapeutic options are more limited and or the prognosis is worse. In such examinations, the status of 158P3D2 in a biological sample of interest can be compared, for example, to the status of 158P3D2 in a corresponding normal sample (e.g. a sample from that individual or alternatively another individual that is not affected by a pathology). An alteration in the status of 158P3D2 in the biological sample (as compared to the normal sample) provides evidence of dysregulated cellular growth. In addition to using a biological sample that is not affected by a pathology as a normal sample, one can also use a predetermined normative value such as a predetermined normal level of mRNA expression (see, e.g., Grever et al., J. Comp. Neurol. 1996 Dec. 9; 376(2): 306-14 and U.S. Pat. No. 5,837,501) to compare 158P3D2 status in a sample.

The term “status” in this context is used according to its art accepted meaning and refers to the condition or state of a gene and its products. Typically, skilled artisans use a number of parameters to evaluate the condition or state of a gene and its products. These include, but are not limited to the location of expressed gene products (including the location of 158P3D2 expressing cells) as well as the level, and biological activity of expressed gene products (such as 158P3D2 mRNA, polynucleotides and polypeptides). Typically, an alteration in the status of 158P3D2 comprises a change in the location of 158P3D2 and/or 158P3D2 expressing cells and/or an increase in 158P3D2 mRNA and/or protein expression.

158P3D2 status in a sample can be analyzed by a number of means well known in the art, including without limitation, immunohistochemical analysis, in situ hybridization, RT-PCR analysis on laser capture micro-dissected samples, Western blot analysis, and tissue array analysis. Typical protocols for evaluating the status of a 158P3D2 gene and gene products are found, for example in Ausubel et al. eds., 1995, Current Protocols In Molecular Biology, Units 2 (Northern Blotting), 4 (Southern Blotting), 15 (Immunoblotting) and 18 (PCR Analysis). Thus, the status of 158P3D2 in a biological sample is evaluated by various methods utilized by skilled artisans including, but not limited to genomic Southern analysis (to examine, for example perturbations in a 158P3D2 gene), Northern analysis and/or PCR analysis of 158P3D2 mRNA (to examine, for example alterations in the polynucleotide sequences or expression levels of 158P3D2 mRNAs), and, Western and/or immunohistochemical analysis (to examine, for example alterations in polypeptide sequences, alterations in polypeptide localization within a sample, alterations in expression levels of 158P3D2 proteins and/or associations of 158P3D2 proteins with polypeptide binding partners). Detectable 158P3D2 polynucleotides include, for example, a 158P3D2 gene or fragment thereof, 158P3D2 mRNA, alternative splice variants, 158P3D2 mRNAs, and recombinant DNA or RNA molecules containing a 158P3D2 polynucleotide.

The expression profile of 158P3D2 makes it a diagnostic marker for local and/or metastasized disease, and provides information on the growth or oncogenic potential of a biological sample. In particular, the status of 158P3D2 provides information useful for predicting susceptibility to particular disease stages, progression, and/or tumor aggressiveness. The invention provides methods and assays for determining 158P3D2 status and diagnosing cancers that express 158P3D2, such as cancers of the tissues listed in Table I. For example, because 158P3D2 mRNA is so highly expressed in prostate and other cancers relative to normal prostate tissue, assays that evaluate the levels of 158P3D2 mRNA transcripts or proteins in a biological sample can be used to diagnose a disease associated with 158P3D2 dysregulation, and can provide prognostic information useful in defining appropriate therapeutic options.

The expression status of 158P3D2 provides information including the presence, stage and location of dysplastic, precancerous and cancerous cells, predicting susceptibility to various stages of disease, and/or for gauging tumor aggressiveness. Moreover, the expression profile makes it useful as an imaging reagent for metastasized disease. Consequently, an aspect of the invention is directed to the various molecular prognostic and diagnostic methods for examining the status of 158P3D2 in biological samples such as those from individuals suffering from, or suspected of suffering from a pathology characterized by dysregulated cellular growth, such as cancer.

As described above, the status of 158P3D2 in a biological sample can be examined by a number of well-known procedures in the art. For example, the status of 158P3D2 in a biological sample taken from a specific location in the body can be examined by evaluating the sample for the presence or absence of 158P3D2 expressing cells (e.g. those that express 158P3D2 mRNAs or proteins). This examination can provide evidence of dysregulated cellular growth, for example, when 158P3D2-expressing cells are found in a biological sample that does not normally contain such cells (such as a lymph node), because such alterations in the status of 158P3D2 in a biological sample are often associated with dysregulated cellular growth. Specifically, one indicator of dysregulated cellular growth is the metastases of cancer cells from an organ of origin (such as the prostate) to a different area of the body (such as a lymph node). In this context, evidence of dysregulated cellular growth is important for example because occult lymph node metastases can be detected in a substantial proportion of patients with prostate cancer, and such metastases are associated with known predictors of disease progression (see, e.g., Murphy et al., Prostate 42(4): 315-317 (2000); Su et al., Semin. Surg. Oncol. 18(1): 17-28 (2000) and Freeman et al., J Urol 1995 August 154(2 Pt 1):474-8).

In one aspect, the invention provides methods for monitoring 158P3D2 gene products by determining the status of 158P3D2 gene products expressed by cells from an individual suspected of having a disease associated with dysregulated cell growth (such as hyperplasia or cancer) and then comparing the status so determined to the status of 158P3D2 gene products in a corresponding normal sample. The presence of aberrant 158P3D2 gene products in the test sample relative to the normal sample provides an indication of the presence of dysregulated cell growth within the cells of the individual.

In another aspect, the invention provides assays useful in determining the presence of cancer in an individual, comprising detecting a significant increase in 158P3D2 mRNA or protein expression in a test cell or tissue sample relative to expression levels in the corresponding normal cell or tissue. The presence of 158P3D2 mRNA can, for example, be evaluated in tissue samples including but not limited to those listed in Table I. The presence of significant 158P3D2 expression in any of these tissues is useful to indicate the emergence, presence and/or severity of a cancer, since the corresponding normal tissues do not express 158P3D2 mRNA or express it at lower levels.

In a related embodiment, 158P3D2 status is determined at the protein level rather than at the nucleic acid level. For example, such a method comprises determining the level of 158P3D2 protein expressed by cells in a test tissue sample and comparing the level so determined to the level of 158P3D2 expressed in a corresponding normal sample. In one embodiment, the presence of 158P3D2 protein is evaluated, for example, using immunohistochemical methods. 158P3D2 antibodies or binding partners capable of detecting 158P3D2 protein expression are used in a variety of assay formats well known in the art for this purpose.

In a further embodiment, one can evaluate the status of 158P3D2 nucleotide and amino acid sequences in a biological sample in order to identify perturbations in the structure of these molecules. These perturbations can include insertions, deletions, substitutions and the like. Such evaluations are useful because perturbations in the nucleotide and amino acid sequences are observed in a large number of proteins associated with a growth dysregulated phenotype (see, e.g., Marrogi et al., 1999, J. Cutan. Pathol. 26(8):369-378). For example, a mutation in the sequence of 158P3D2 may be indicative of the presence or promotion of a tumor. Such assays therefore have diagnostic and predictive value where a mutation in 158P3D2 indicates a potential loss of function or increase in tumor growth.

A wide variety of assays for observing perturbations in nucleotide and amino acid sequences are well known in the art. For example, the size and structure of nucleic acid or amino acid sequences of 158P3D2 gene products are observed by the Northern, Southern, Western, PCR and DNA sequencing protocols discussed herein. In addition, other methods for observing perturbations in nucleotide and amino acid sequences such as single strand conformation polymorphism analysis are well known in the art (see, e.g., U.S. Pat. Nos. 5,382,510 issued 7 Sep. 1999, and 5,952,170 issued 17 Jan. 1995).

Additionally, one can examine the methylation status of a 158P3D2 gene in a biological sample. Aberrant demethylation and/or hypermethylation of CpG islands in gene 5′ regulatory regions frequently occurs in immortalized and transformed cells, and can result in altered expression of various genes. For example, promoter hypermethylation of the pi-class glutathione S-transferase (a protein expressed in normal prostate but not expressed in >90% of prostate carcinomas) appears to permanently silence transcription of this gene and is the most frequently detected genomic alteration in prostate carcinomas (De Marzo et al., Am. J. Pathol. 155(6): 1985-1992 (1999)). In addition, this alteration is present in at least 70% of cases of high-grade prostatic intraepithelial neoplasia (PIN) (Brooks et al., Cancer Epidemiol. Biomarkers Prev., 1998, 7:531-536). In another example, expression of the LAGE-I tumor specific gene (which is not expressed in normal prostate but is expressed in 25-50% of prostate cancers) is induced by deoxy-azacytidine in lymphoblastoid cells, suggesting that tumoral expression is due to demethylation (Lethe et al., Int. J. Cancer 76(6): 903-908 (1998)). A variety of assays for examining methylation status of a gene are well known in the art. For example, one can utilize, in Southern hybridization approaches, methylation-sensitive restriction enzymes that cannot cleave sequences that contain methylated CpG sites to assess the methylation status of CpG islands. In addition, MSP (methylation specific PCR) can rapidly profile the methylation status of all the CpG sites present in a CpG island of a given gene. This procedure involves initial modification of DNA by sodium bisulfite (which will convert all unmethylated cytosines to uracil) followed by amplification using primers specific for methylated versus unmethylated DNA. Protocols involving methylation interference can also be found for example in Current Protocols In Molecular Biology, Unit 12, Frederick M. Ausubel et al. eds., 1995.

Gene amplification is an additional method for assessing the status of 158P3D2. Gene amplification is measured in a sample directly, for example, by conventional Southern blotting or Northern blotting to quantitate the transcription of mRNA (Thomas, 1980, Proc. Natl. Acad. Sci. USA, 77:5201-5205), dot blotting (DNA analysis), or in situ hybridization, using an appropriately labeled probe, based on the sequences provided herein. Alternatively, antibodies are employed that recognize specific duplexes, including DNA duplexes, RNA duplexes, and DNA-RNA hybrid duplexes or DNA-protein duplexes. The antibodies in turn are labeled and the assay carried out where the duplex is bound to a surface, so that upon the formation of duplex on the surface, the presence of antibody bound to the duplex can be detected.

Biopsied tissue or peripheral blood can be conveniently assayed for the presence of cancer cells using for example, Northern, dot blot or RT-PCR analysis to detect 158P3D2 expression. The presence of RT-PCR amplifiable 158P3D2 mRNA provides an indication of the presence of cancer. RT-PCR assays are well known in the art. RT-PCR detection assays for tumor cells in peripheral blood are currently being evaluated for use in the diagnosis and management of a number of human solid tumors. In the prostate cancer field, these include RT-PCR assays for the detection of cells expressing PSA and PSM (Verkaik et al., 1997, Urol. Res. 25:373-384; Ghossein et al., 1995, J. Clin. Oncol. 13:1195-2000; Heston et al., 1995, Clin. Chem. 41:1687-1688).

A further aspect of the invention is an assessment of the susceptibility that an individual has for developing cancer. In one embodiment, a method for predicting susceptibility to cancer comprises detecting 158P3D2 mRNA or 158P3D2 protein in a tissue sample, its presence indicating susceptibility to cancer, wherein the degree of 158P3D2 mRNA expression correlates to the degree of susceptibility. In a specific embodiment, the presence of 158P3D2 in prostate or other tissue is examined, with the presence of 158P3D2 in the sample providing an indication of prostate cancer susceptibility (or the emergence or existence of a prostate tumor). Similarly, one can evaluate the integrity 158P3D2 nucleotide and amino acid sequences in a biological sample, in order to identify perturbations in the structure of these molecules such as insertions, deletions, substitutions and the like. The presence of one or more perturbations in 158P3D2 gene products in the sample is an indication of cancer susceptibility (or the emergence or existence of a tumor).

The invention also comprises methods for gauging tumor aggressiveness. In one embodiment, a method for gauging aggressiveness of a tumor comprises determining the level of 158P3D2 mRNA or 158P3D2 protein expressed by tumor cells, comparing the level so determined to the level of 158P3D2 mRNA or 158P3D2 protein expressed in a corresponding normal tissue taken from the same individual or a normal tissue reference sample, wherein the degree of 158P3D2 mRNA or 158P3D2 protein expression in the tumor sample relative to the normal sample indicates the degree of aggressiveness. In a specific embodiment, aggressiveness of a tumor is evaluated by determining the extent to which 158P3D2 is expressed in the tumor cells, with higher expression levels indicating more aggressive tumors. Another embodiment is the evaluation of the integrity of 158P3D2 nucleotide and amino acid sequences in a biological sample, in order to identify perturbations in the structure of these molecules such as insertions, deletions, substitutions and the like. The presence of one or more perturbations indicates more aggressive tumors.

Another embodiment of the invention is directed to methods for observing the progression of a malignancy in an individual over time. In one embodiment, methods for observing the progression of a malignancy in an individual over time comprise determining the level of 158P3D2 mRNA or 158P3D2 protein expressed by cells in a sample of the tumor, comparing the level so determined to the level of 158P3D2 mRNA or 158P3D2 protein expressed in an equivalent tissue sample taken from the same individual at a different time, wherein the degree of 158P3D2 mRNA or 158P3D2 protein expression in the tumor sample over time provides information on the progression of the cancer. In a specific embodiment, the progression of a cancer is evaluated by determining 158P3D2 expression in the tumor cells over time, where increased expression over time indicates a progression of the cancer. Also, one can evaluate the integrity 158P3D2 nucleotide and amino acid sequences in a biological sample in order to identify perturbations in the structure of these molecules such as insertions, deletions, substitutions and the like, where the presence of one or more perturbations indicates a progression of the cancer.

The above diagnostic approaches can be combined with any one of a wide variety of prognostic and diagnostic protocols known in the art. For example, another embodiment of the invention is directed to methods for observing a coincidence between the expression of 158P3D2 gene and 158P3D2 gene products (or perturbations in 158P3D2 gene and 158P3D2 gene products) and a factor that is associated with malignancy, as a means for diagnosing and prognosticating the status of a tissue sample. A wide variety of factors associated with malignancy can be utilized, such as the expression of genes associated with malignancy (e.g. PSA, PSCA and PSM expression for prostate cancer etc.) as well as gross cytological observations (see, e.g., Bocking et al., 1984, Anal. Quant. Cytol. 6(2):74-88; Epstein, 1995, Hum. Pathol. 26(2):223-9; Thorson et al., 1998, Mod. Pathol. 11(6):543-51; Baisden et al., 1999, Am. J. Surg. Pathol. 23(8):918-24). Methods for observing a coincidence between the expression of 158P3D2 gene and 158P3D2 gene products (or perturbations in 158P3D2 gene and 158P3D2 gene products) and another factor that is associated with malignancy are useful, for example, because the presence of a set of specific factors that coincide with disease provides information crucial for diagnosing and prognosticating the status of a tissue sample.

In one embodiment, methods for observing a coincidence between the expression of 158P3D2 gene and 158P3D2 gene products (or perturbations in 158P3D2 gene and 158P3D2 gene products) and another factor associated with malignancy entails detecting the overexpression of 158P3D2 mRNA or protein in a tissue sample, detecting the overexpression of PSA mRNA or protein in a tissue sample (or PSCA or PSM expression), and observing a coincidence of 158P3D2 mRNA or protein and PSA mRNA or protein overexpression (or PSCA or PSM expression). In a specific embodiment, the expression of 158P3D2 and PSA mRNA in prostate tissue is examined, where the coincidence of 158P3D2 and PSA mRNA overexpression in the sample indicates the existence of prostate cancer, prostate cancer susceptibility or the emergence or status of a prostate tumor.

Methods for detecting and quantifying the expression of 158P3D2 mRNA or protein are described herein, and standard nucleic acid and protein detection and quantification technologies are well known in the art. Standard methods for the detection and quantification of 158P3D2 mRNA include in situ hybridization using labeled 158P3D2 riboprobes, Northern blot and related techniques using 158P3D2 polynucleotide probes, RT-PCR analysis using primers specific for 158P3D2, and other amplification type detection methods, such as, for example, branched DNA, SISBA, TMA and the like. In a specific embodiment, semi-quantitative RT-PCR is used to detect and quantify 158P3D2 mRNA expression. Any number of primers capable of amplifying 158P3D2 can be used for this purpose, including but not limited to the various primer sets specifically described herein. In a specific embodiment, polyclonal or monoclonal antibodies specifically reactive with the wild-type 158P3D2 protein can be used in an immunohistochemical assay of biopsied tissue.

IX.) IDENTIFICATION OF MOLECULES THAT INTERACT WITH 158P3D2

The 158P3D2 protein and nucleic acid sequences disclosed herein allow a skilled artisan to identify proteins, small molecules and other agents that interact with 158P3D2, as well as pathways activated by 158P3D2 via any one of a variety of art accepted protocols. For example, one can utilize one of the so-called interaction trap systems (also referred to as the “two-hybrid assay”). In such systems, molecules interact and reconstitute a transcription factor which directs expression of a reporter gene, whereupon the expression of the reporter gene is assayed. Other systems identify protein-protein interactions in vivo through reconstitution of a eukaryotic transcriptional activator, see, e.g., U.S. Pat. Nos. 5,955,280 issued 21 Sep. 1999, 5,925,523 issued 20 Jul. 1999, 5,846,722 issued 8 Dec. 1998 and 6,004,746 issued 21 Dec. 1999. Algorithms are also available in the art for genome-based predictions of protein function (see, e.g., Marcotte, et al., Nature 402: 4 Nov. 1999, 83-86).

Alternatively one can screen peptide libraries to identify molecules that interact with 158P3D2 protein sequences. In such methods, peptides that bind to 158P3D2 are identified by screening libraries that encode a random or controlled collection of amino acids. Peptides encoded by the libraries are expressed as fusion proteins of bacteriophage coat proteins, the bacteriophage particles are then screened against the 158P3D2 protein(s).

Accordingly, peptides having a wide variety of uses, such as therapeutic, prognostic or diagnostic reagents, are thus identified without any prior information on the structure of the expected ligand or receptor molecule. Typical peptide libraries and screening methods that can be used to identify molecules that interact with 158P3D2 protein sequences are disclosed for example in U.S. Pat. Nos. 5,723,286 issued 3 Mar. 1998 and 5,733,731 issued 31 Mar. 1998.

Alternatively, cell lines that express 158P3D2 are used to identify protein-protein interactions mediated by 158P3D2. Such interactions can be examined using immunoprecipitation techniques (see, e.g., Hamilton B. J., et al. Biochem. Biophys. Res. Commun. 1999, 261:646-51). 158P3D2 protein can be immunoprecipitated from 158P3D2-expressing cell lines using anti-158P3D2 antibodies. Alternatively, antibodies against His-tag can be used in a cell line engineered to express fusions of 158P3D2 and a His-tag (vectors mentioned above). The immunoprecipitated complex can be examined for protein association by procedures such as Western blotting, ³⁵S-methionine labeling of proteins, protein microsequencing, silver staining and two-dimensional gel electrophoresis.

Small molecules and ligands that interact with 158P3D2 can be identified through related embodiments of such screening assays. For example, small molecules can be identified that interfere with protein function, including molecules that interfere with 158P3D2's ability to mediate phosphorylation and de-phosphorylation, interaction with DNA or RNA molecules as an indication of regulation of cell cycles, second messenger signaling or tumorigenesis. Similarly, small molecules that modulate 158P3D2-related ion channel, protein pump, or cell communication functions are identified and used to treat patients that have a cancer that expresses 158P3D2 (see, e.g., Hille, B., Ionic Channels of Excitable Membranes 2^(nd) Ed., Sinauer Assoc., Sunderland, Mass., 1992). Moreover, ligands that regulate 158P3D2 function can be identified based on their ability to bind 158P3D2 and activate a reporter construct. Typical methods are discussed for example in U.S. Pat. No. 5,928,868 issued 27 Jul. 1999, and include methods for forming hybrid ligands in which at least one ligand is a small molecule. In an illustrative embodiment, cells engineered to express a fusion protein of 158P3D2 and a DNA-binding protein are used to co-express a fusion protein of a hybrid ligand/small molecule and a cDNA library transcriptional activator protein. The cells further contain a reporter gene, the expression of which is conditioned on the proximity of the first and second fusion proteins to each other, an event that occurs only if the hybrid ligand binds to target sites on both hybrid proteins. Those cells that express the reporter gene are selected and the unknown small molecule or the unknown ligand is identified. This method provides a means of identifying modulators which activate or inhibit 158P3D2.

An embodiment of this invention comprises a method of screening for a molecule that interacts with an 158P3D2 amino acid sequence shown in FIG. 2 or FIG. 3, comprising the steps of contacting a population of molecules with a 158P3D2 amino acid sequence, allowing the population of molecules and the 158P3D2 amino acid sequence to interact under conditions that facilitate an interaction, determining the presence of a molecule that interacts with the 158P3D2 amino acid sequence, and then separating molecules that do not interact with the 158P3D2 amino acid sequence from molecules that do. In a specific embodiment, the method further comprises purifying, characterizing and identifying a molecule that interacts with the 158P3D2 amino acid sequence. The identified molecule can be used to modulate a function performed by 158P3D2. In a preferred embodiment, the 158P3D2 amino acid sequence is contacted with a library of peptides.

X.) THERAPEUTIC METHODS AND COMPOSITIONS

The identification of 158P3D2 as a protein that is normally expressed in a restricted set of tissues, but which is also expressed in prostate and other cancers, opens a number of therapeutic approaches to the treatment of such cancers. As contemplated herein, 158P3D2 functions as a transcription factor involved in activating tumor-promoting genes or repressing genes that block tumorigenesis.

Accordingly, therapeutic approaches that inhibit the activity of a 158P3D2 protein are useful for patients suffering from a cancer that expresses 158P3D2. These therapeutic approaches generally fall into two classes. One class comprises various methods for inhibiting the binding or association of a 158P3D2 protein with its binding partner or with other proteins. Another class comprises a variety of methods for inhibiting the transcription of a 158P3D2 gene or translation of 158P3D2 mRNA.

X.A.) Anti-Cancer Vaccines

The invention provides cancer vaccines comprising a 158P3D2-related protein or 158P3D2-related nucleic acid. In view of the expression of 158P3D2, cancer vaccines prevent and/or treat 158P3D2-expressing cancers with minimal or no effects on non-target tissues. The use of a tumor antigen in a vaccine that generates humoral and/or cell-mediated immune responses as anti-cancer therapy is well known in the art and has been employed in prostate cancer using human PSMA and rodent PAP immunogens (Hodge et al., 1995, Int. J. Cancer 63:231-237; Fong et al., 1997, J. Immunol. 159:3113-3117).

Such methods can be readily practiced by employing a 158P3D2-related protein, or an 158P3D2-encoding nucleic acid molecule and recombinant vectors capable of expressing and presenting the 158P3D2 immunogen (which typically comprises a number of antibody or T cell epitopes). Skilled artisans understand that a wide variety of vaccine systems for delivery of immunoreactive epitopes are known in the art (see, e.g., Heryln et al., Ann Med 1999 Feb. 31(1):66-78; Maruyama et al., Cancer Immunol Immunother 2000 June 49(3):123-32) Briefly, such methods of generating an immune response (e.g. humoral and/or cell-mediated) in a mammal, comprise the steps of: exposing the mammal's immune system to an immunoreactive epitope (e.g. an epitope present in a 158P3D2 protein shown in FIG. 3 or analog or homolog thereof) so that the mammal generates an immune response that is specific for that epitope (e.g. generates antibodies that specifically recognize that epitope). In a preferred method, a 158P3D2 immunogen contains a biological motif, see e.g., Tables V-XIX, or a peptide of a size range from 158P3D2 indicated in FIG. 5, FIG. 6, FIG. 7, FIG. 8, and FIG. 9.

The entire 158P3D2 protein, immunogenic regions or epitopes thereof can be combined and delivered by various means. Such vaccine compositions can include, for example, lipopeptides (e.g., Vitiello, A. et al., J. Clin. Invest. 95:341, 1995), peptide compositions encapsulated in poly(DL-lactide-co-glycolide) (“PLG”) microspheres (see, e.g., Eldridge, et al., Molec. Immunol. 28:287-294, 1991: Alonso et al., Vaccine 12:299-306, 1994; Jones et al., Vaccine 13:675-681, 1995), peptide compositions contained in immune stimulating complexes (ISCOMS) (see, e.g., Takahashi et al., Nature 344:873-875, 1990; Hu et al., Clin Exp Immunol. 113:235-243, 1998), multiple antigen peptide systems (MAPs) (see e.g., Tam, J. P., Proc. Natl. Acad. Sci. U.S.A. 85:5409-5413, 1988; Tam, J. P., J. Immunol. Methods 196:17-32, 1996), peptides formulated as multivalent peptides; peptides for use in ballistic delivery systems, typically crystallized peptides, viral delivery vectors (Perkus, M. E. et al., In: Concepts in vaccine development, Kaufmann, S. H. E., ed., p. 379, 1996; Chakrabarti, S. et al., Nature 320:535, 1986; Hu, S. L. et al., Nature 320:537, 1986; Kieny, M.-P. et al., AIDS Bio/Technology 4:790, 1986; Top, F. H. et al., J. Infect. Dis. 124:148, 1971; Chanda, P. K. et al., Virology 175:535, 1990), particles of viral or synthetic origin (e.g., Kofler, N. et al., J. Immunol. Methods. 192:25, 1996; Eldridge, J. H. et al., Sem. Hematol. 30:16, 1993; Falo, L. D., Jr. et al., Nature Med 7:649, 1995), adjuvants (Warren, H. S., Vogel, F. R., and Chedid, L. A. Annu. Rev. Immunol. 4:369, 1986; Gupta, R. K. et al., Vaccine 11:293, 1993), liposomes (Reddy, R. et al., J. Immunol 148:1585, 1992; Rock, K. L., Immunol. Today 17:131, 1996), or, naked or particle absorbed cDNA (Ulmer, J. B. et al., Science 259:1745, 1993; Robinson, H. L., Hunt, L. A., and Webster, R. G., Vaccine 11:957, 1993; Shiver, J. W. et al., In: Concepts in vaccine development, Kaufmann, S. H. E., ed., p. 423, 1996; Cease, K. B., and Berzofsky, J. A., Annu. Rev. Immunol. 12:923, 1994 and Eldridge, J. H. et al., Sem. Hematol. 30:16, 1993). Toxin-targeted delivery technologies, also known as receptor mediated targeting, such as those of Avant Immunotherapeutics, Inc. (Needham, Mass.) may also be used.

In patients with 158P3D2-associated cancer, the vaccine compositions of the invention can also be used in conjunction with other treatments used for cancer, e.g., surgery, chemotherapy, drug therapies, radiation therapies, etc. including use in combination with immune adjuvants such as IL-2, IL-12, GM-CSF, and the like.

Cellular Vaccines:

CTL epitopes can be determined using specific algorithms to identify peptides within 158P3D2 protein that bind corresponding HLA alleles (see e.g., Table IV; Epimer™ and Epimatrix™, Brown University (URL www.brown.edu/Research/TB-HIV_Lab/epimatrix/epimatrix.html); and, BIMAS, (URL bimas.dcrt.nih.gov/; SYFPEITHI at URL syfpeithi.bmi-heidelberg.com/). In a preferred embodiment, a 158P3D2 immunogen contains one or more amino acid sequences identified using techniques well known in the art, such as the sequences shown in Tables V-XIX or a peptide of 8, 9, 10 or 11 amino acids specified by an HLA Class I motif/supermotif (e.g., Table IV (A), Table IV (D), or Table IV (E)) and/or a peptide of at least 9 amino acids that comprises an HLA Class II motif/supermotif (e.g., Table IV (B) or Table IV (C)). As is appreciated in the art, the HLA Class I binding groove is essentially closed ended so that peptides of only a particular size range can fit into the groove and be bound, generally HLA Class I epitopes are 8, 9, 10, or 11 amino acids long. In contrast, the HLA Class II binding groove is essentially open ended; therefore a peptide of about 9 or more amino acids can be bound by an HLA Class II molecule. Due to the binding groove differences between HLA Class I and II, HLA Class I motifs are length specific, i.e., position two of a Class I motif is the second amino acid in an amino to carboxyl direction of the peptide. The amino acid positions in a Class II motif are relative only to each other, not the overall peptide, i.e., additional amino acids can be attached to the amino and/or carboxyl termini of a motif-bearing sequence. HLA Class II epitopes are often 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, or 25 amino acids long, or longer than 25 amino acids.

Antibody-Based Vaccines

A wide variety of methods for generating an immune response in a mammal are known in the art (for example as the first step in the generation of hybridomas). Methods of generating an immune response in a mammal comprise exposing the mammal's immune system to an immunogenic epitope on a protein (e.g. a 158P3D2 protein) so that an immune response is generated. A typical embodiment consists of a method for generating an immune response to 158P3D2 in a host, by contacting the host with a sufficient amount of at least one 158P3D2 B cell or cytotoxic T-cell epitope or analog thereof; and at least one periodic interval thereafter re-contacting the host with the 158P3D2 B cell or cytotoxic T-cell epitope or analog thereof. A specific embodiment consists of a method of generating an immune response against a 158P3D2-related protein or a man-made multiepitopic peptide comprising: administering 158P3D2 immunogen (e.g. a 158P3D2 protein or a peptide fragment thereof, an 158P3D2 fusion protein or analog etc.) in a vaccine preparation to a human or another mammal. Typically, such vaccine preparations further contain a suitable adjuvant (see, e.g., U.S. Pat. No. 6,146,635) or a universal helper epitope such as a PADRE™ peptide (Epimmune Inc., San Diego, Calif.; see, e.g., Alexander et al., J. Immunol. 2000 164(3); 164(3): 1625-1633; Alexander et al., Immunity 1994 1(9): 751-761 and Alexander et al., Immunol. Res. 1998 18(2): 79-92). An alternative method comprises generating an immune response in an individual against a 158P3D2 immunogen by: administering in vivo to muscle or skin of the individual's body a DNA molecule that comprises a DNA sequence that encodes an 158P3D2 immunogen, the DNA sequence operatively linked to regulatory sequences which control the expression of the DNA sequence; wherein the DNA molecule is taken up by cells, the DNA sequence is expressed in the cells and an immune response is generated against the immunogen (see, e.g., U.S. Pat. No. 5,962,428). Optionally a genetic vaccine facilitator such as anionic lipids; saponins; lectins; estrogenic compounds; hydroxylated lower alkyls; dimethyl sulfoxide; and urea is also administered. In addition, an antiidiotypic antibody can be administered that mimics 158P3D2, in order to generate a response to the target antigen.

Nucleic Acid Vaccines:

Vaccine compositions of the invention include nucleic acid-mediated modalities. DNA or RNA that encode protein(s) of the invention can be administered to a patient. Genetic immunization methods can be employed to generate prophylactic or therapeutic humoral and cellular immune responses directed against cancer cells expressing 158P3D2. Constructs comprising DNA encoding a 158P3D2-related protein/immunogen and appropriate regulatory sequences can be injected directly into muscle or skin of an individual, such that the cells of the muscle or skin take-up the construct and express the encoded 158P3D2 protein/immunogen. Alternatively, a vaccine comprises a 158P3D2-related protein. Expression of the 158P3D2-related protein immunogen results in the generation of prophylactic or therapeutic humoral and cellular immunity against cells that bear a 158P3D2 protein. Various prophylactic and therapeutic genetic immunization techniques known in the art can be used. Nucleic acid-based delivery is described, for instance, in Wolff et. al., Science 247:1465 (1990) as well as U.S. Pat. Nos. 5,580,859; 5,589,466; 5,804,566; 5,739,118; 5,736,524; 5,679,647; WO 98/04720. Examples of DNA-based delivery technologies include “naked DNA”, facilitated (bupivicaine, polymers, peptide-mediated) delivery, cationic lipid complexes, and particle-mediated (“gene gun”) or pressure-mediated delivery (see, e.g., U.S. Pat. No. 5,922,687).

For therapeutic or prophylactic immunization purposes, proteins of the invention can be expressed via viral or bacterial vectors. Various viral gene delivery systems that can be used in the practice of the invention include, but are not limited to, vaccinia, fowipox, canarypox, adenovirus, influenza, poliovirus, adeno-associated virus, lentivirus, and sindbis virus (see, e.g., Restifo, 1996, Curr. Opin. Immunol. 8:658-663; Tsang et al. J. Natl. Cancer Inst. 87:982-990 (1995)). Non-viral delivery systems can also be employed by introducing naked DNA encoding a 158P3D2-related protein into the patient (e.g., intramuscularly or intradermally) to induce an anti-tumor response.

Vaccinia virus is used, for example, as a vector to express nucleotide sequences that encode the peptides of the invention. Upon introduction into a host, the recombinant vaccinia virus expresses the protein immunogenic peptide, and thereby elicits a host immune response. Vaccinia vectors and methods useful in immunization protocols are described in, e.g., U.S. Pat. No. 4,722,848. Another vector is BCG (Bacille Calmette Guerin). BCG vectors are described in Stover et al., Nature 351:456-460 (1991). A wide variety of other vectors useful for therapeutic administration or immunization of the peptides of the invention, e.g. adeno and adeno-associated virus vectors, retroviral vectors, Salmonella typhi vectors, detoxified anthrax toxin vectors, and the like, will be apparent to those skilled in the art from the description herein.

Thus, gene delivery systems are used to deliver a 158P3D2-related nucleic acid molecule. In one embodiment, the full-length human 158P3D2 cDNA is employed. In another embodiment, 158P3D2 nucleic acid molecules encoding specific cytotoxic T lymphocyte (CTL) and/or antibody epitopes are employed.

Ex Vivo Vaccines

Various ex vivo strategies can also be employed to generate an immune response. One approach involves the use of antigen presenting cells (APCs) such as dendritic cells (DC) to present 158P3D2 antigen to a patient's immune system. Dendritic cells express MHC class I and II molecules, B7 co-stimulator, and IL-12, and are thus highly specialized antigen presenting cells. In prostate cancer, autologous dendritic cells pulsed with peptides of the prostate-specific membrane antigen (PSMA) are being used in a Phase I clinical trial to stimulate prostate cancer patients' immune systems (Tjoa et al., 1996, Prostate 28:65-69; Murphy et al., 1996, Prostate 29:371-380). Thus, dendritic cells can be used to present 158P3D2 peptides to T cells in the context of MHC class I or II molecules. In one embodiment, autologous dendritic cells are pulsed with 158P3D2 peptides capable of binding to MHC class I and/or class II molecules. In another embodiment, dendritic cells are pulsed with the complete 158P3D2 protein. Yet another embodiment involves engineering the overexpression of a 158P3D2 gene in dendritic cells using various implementing vectors known in the art, such as adenovirus (Arthur et al., 1997, Cancer Gene Ther. 4:17-25), retrovirus (Henderson et al., 1996, Cancer Res. 56:3763-3770), lentivirus, adeno-associated virus, DNA transfection (Ribas et al., 1997, Cancer Res. 57:2865-2869), or tumor-derived RNA transfection (Ashley et al., 1997, J. Exp. Med. 186:1177-1182). Cells that express 158P3D2 can also be engineered to express immune modulators, such as GM-CSF, and used as immunizing agents.

X.B.) 158P3D2 as a Target for Antibody-Based Therapy

158P3D2 is an attractive target for antibody-based therapeutic strategies. A number of antibody strategies are known in the art for targeting both extracellular and intracellular molecules (see, e.g., complement and ADCC mediated killing as well as the use of intrabodies). Because 158P3D2 is expressed by cancer cells of various lineages relative to corresponding normal cells, systemic administration of 158P3D2-immunoreactive compositions are prepared that exhibit excellent sensitivity without toxic, non-specific and/or non-target effects caused by binding of the immunoreactive composition to non-target organs and tissues. Antibodies specifically reactive with domains of 158P3D2 are useful to treat 158P3D2-expressing cancers systemically, either as conjugates with a toxin or therapeutic agent, or as naked antibodies capable of inhibiting cell proliferation or function.

158P3D2 antibodies can be introduced into a patient such that the antibody binds to 158P3D2 and modulates a function, such as an interaction with a binding partner, and consequently mediates destruction of the tumor cells and/or inhibits the growth of the tumor cells. Mechanisms by which such antibodies exert a therapeutic effect can include complement-mediated cytolysis, antibody-dependent cellular cytotoxicity, modulation of the physiological function of 158P3D2, inhibition of ligand binding or signal transduction pathways, modulation of tumor cell differentiation, alteration of tumor angiogenesis factor profiles, and/or apoptosis.

Those skilled in the art understand that antibodies can be used to specifically target and bind immunogenic molecules such as an immunogenic region of a 158P3D2 sequence shown in FIG. 2 or FIG. 3. In addition, skilled artisans understand that it is routine to conjugate antibodies to cytotoxic agents (see, e.g., Slevers et al. Blood 93:11 3678-3684 (Jun. 1, 1999)). When cytotoxic and/or therapeutic agents are delivered directly to cells, such as by conjugating them to antibodies specific for a molecule expressed by that cell (e.g. 158P3D2), the cytotoxic agent will exert its known biological effect (i.e. cytotoxicity) on those cells.

A wide variety of compositions and methods for using antibody-cytotoxic agent conjugates to kill cells are known in the art. In the context of cancers, typical methods entail administering to an animal having a tumor a biologically effective amount of a conjugate comprising a selected cytotoxic and/or therapeutic agent linked to a targeting agent (e.g. an anti-158P3D2 antibody) that binds to a marker (e.g. 158P3D2) expressed, accessible to binding or localized on the cell surfaces. A typical embodiment is a method of delivering a cytotoxic and/or therapeutic agent to a cell expressing 158P3D2, comprising conjugating the cytotoxic agent to an antibody that immunospecifically binds to a 158P3D2 epitope, and, exposing the cell to the antibody-agent conjugate. Another illustrative embodiment is a method of treating an individual suspected of suffering from metastasized cancer, comprising a step of administering parenterally to said individual a pharmaceutical composition comprising a therapeutically effective amount of an antibody conjugated to a cytotoxic and/or therapeutic agent.

Cancer immunotherapy using anti-158P3D2 antibodies can be done in accordance with various approaches that have been successfully employed in the treatment of other types of cancer, including but not limited to colon cancer (Arlen et al., 1998, Crit. Rev. Immunol. 18:133-138), multiple myeloma (Ozaki et al., 1997, Blood 90:3179-3186, Tsunenari et al., 1997, Blood 90:2437-2444), gastric cancer (Kasprzyk et al., 1992, Cancer Res. 52:2771-2776), B-cell lymphoma (Funakoshi et al., 1996, J. Immunother. Emphasis Tumor Immunol. 19:93-101), leukemia (Zhong et al., 1996, Leuk. Res. 20:581-589), colorectal cancer (Moun et al., 1994, Cancer Res. 54:6160-6166; Velders et al., 1995, Cancer Res. 55:4398-4403), and breast cancer (Shepard et al., 1991, J. Clin. Immunol. 11:117-127). Some therapeutic approaches involve conjugation of naked antibody to a toxin or radioisotope, such as the conjugation of Y⁹¹ or I¹³¹ to anti-CD20 antibodies (e.g., Zevalin™, IDEC Pharmaceuticals Corp. or Bexxarm, Coulter Pharmaceuticals), while others involve co-administration of antibodies and other therapeutic agents, such as Herceptin™ (trastuzumab) with paclitaxel (Genentech, Inc.). The antibodies can be conjugated to a therapeutic agent. To treat prostate cancer, for example, 158P3D2 antibodies can be administered in conjunction with radiation, chemotherapy or hormone ablation. Also, antibodies can be conjugated to a toxin such as calicheamicin (e.g., Mylotarg™, Wyeth-Ayerst, Madison, N.J., a recombinant humanized IgG₄ kappa antibody conjugated to antitumor antibiotic calicheamicin) or a maytansinoid (e.g., taxane-based Tumor-Activated Prodrug, TAP, platform, ImmunoGen, Cambridge, Mass., also see e.g., U.S. Pat. No. 5,416,064).

Although 158P3D2 antibody therapy is useful for all stages of cancer, antibody therapy can be particularly appropriate in advanced or metastatic cancers. Treatment with the antibody therapy of the invention is indicated for patients who have received one or more rounds of chemotherapy. Alternatively, antibody therapy of the invention is combined with a chemotherapeutic or radiation regimen for patients who have not received chemotherapeutic treatment. Additionally, antibody therapy can enable the use of reduced dosages of concomitant chemotherapy, particularly for patients who do not tolerate the toxicity of the chemotherapeutic agent very well. Fan et al. (Cancer Res. 53:4637-4642, 1993), Prewett et al. (International J. of Onco. 9:217-224, 1996), and Hancock et al. (Cancer Res. 51:4575-4580, 1991) describe the use of various antibodies together with chemotherapeutic agents.

Although 158P3D2 antibody therapy is useful for all stages of cancer, antibody therapy can be particularly appropriate in advanced or metastatic cancers. Treatment with the antibody therapy of the invention is indicated for patients who have received one or more rounds of chemotherapy. Alternatively, antibody therapy of the invention is combined with a chemotherapeutic or radiation regimen for patients who have not received chemotherapeutic treatment. Additionally, antibody therapy can enable the use of reduced dosages of concomitant chemotherapy, particularly for patients who do not tolerate the toxicity of the chemotherapeutic agent very well.

Cancer patients can be evaluated for the presence and level of 158P3D2 expression, preferably using immunohistochemical assessments of tumor tissue, quantitative 158P3D2 imaging, or other techniques that reliably indicate the presence and degree of 158P3D2 expression. Immunohistochemical analysis of tumor biopsies or surgical specimens is preferred for this purpose. Methods for immunohistochemical analysis of tumor tissues are well known in the art.

Anti-158P3D2 monoclonal antibodies that treat prostate and other cancers include those that initiate a potent immune response against the tumor or those that are directly cytotoxic. In this regard, anti-158P3D2 monoclonal antibodies (mAbs) can elicit tumor cell lysis by either complement-mediated or antibody-dependent cell cytotoxicity (ADCC) mechanisms, both of which require an intact Fc portion of the immunoglobulin molecule for interaction with effector cell Fc receptor sites on complement proteins. In addition, anti-158P3D2 mAbs that exert a direct biological effect on tumor growth are useful to treat cancers that express 158P3D2. Mechanisms by which directly cytotoxic mAbs act include: inhibition of cell growth, modulation of cellular differentiation, modulation of tumor angiogenesis factor profiles, and the induction of apoptosis. The mechanism(s) by which a particular anti-158P3D2 mAb exerts an anti-tumor effect is evaluated using any number of in vitro assays that evaluate cell death such as ADCC, ADMMC, complement-mediated cell lysis, and so forth, as is generally known in the art.

In some patients, the use of murine or other non-human monoclonal antibodies, or human/mouse chimeric mAbs can induce moderate to strong immune responses against the non-human antibody. This can result in clearance of the antibody from circulation and reduced efficacy. In the most severe cases, such an immune response can lead to the extensive formation of immune complexes which, potentially, can cause renal failure. Accordingly, preferred monoclonal antibodies used in the therapeutic methods of the invention are those that are either fully human or humanized and that bind specifically to the target 158P3D2 antigen with high affinity but exhibit low or no antigenicity in the patient.

Therapeutic methods of the invention contemplate the administration of single anti-158P3D2 mAbs as well as combinations, or cocktails, of different mAbs. Such mAb cocktails can have certain advantages inasmuch as they contain mAbs that target different epitopes, exploit different effector mechanisms or combine directly cytotoxic mAbs with mAbs that rely on immune effector functionality. Such mAbs in combination can exhibit synergistic therapeutic effects. In addition, anti-158P3D2 mAbs can be administered concomitantly with other therapeutic modalities, including but not limited to various chemotherapeutic agents, androgen-blockers, immune modulators (e.g., IL-2, GM-CSF), surgery or radiation. The anti-158P3D2 mAbs are administered in their “naked” or unconjugated form, or can have a therapeutic agent(s) conjugated to them.

Anti-158P3D2 antibody formulations are administered via any route capable of delivering the antibodies to a tumor cell. Routes of administration include, but are not limited to, intravenous, intraperitoneal, intramuscular, intratumor, intradermal, and the like. Treatment generally involves repeated administration of the anti-158P3D2 antibody preparation, via an acceptable route of administration such as intravenous injection (IV), typically at a dose in the range of about 0.1, 0.2, 0.3, 0.4, 0.5, 0.6, 0.7, 0.8, 0.9, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 15, 20, or 25 mg/kg body weight. In general, doses in the range of 10-1000 mg mAb per week are effective and well tolerated.

Based on clinical experience with the Herceptin™ mAb in the treatment of metastatic breast cancer, an initial loading dose of approximately 4 mg/kg patient body weight IV, followed by weekly doses of about 2 mg/kg IV of the anti-158P3D2 mAb preparation represents an acceptable dosing regimen. Preferably, the initial loading dose is administered as a 90 minute or longer infusion. The periodic maintenance dose is administered as a 30 minute or longer infusion, provided the initial dose was well tolerated. As appreciated by those of skill in the art, various factors can influence the ideal dose regimen in a particular case. Such factors include, for example, the binding affinity and half life of the Ab or mAbs used, the degree of 158P3D2 expression in the patient, the extent of circulating shed 158P3D2 antigen, the desired steady-state antibody concentration level, frequency of treatment, and the influence of chemotherapeutic or other agents used in combination with the treatment method of the invention, as well as the health status of a particular patient.

Optionally, patients should be evaluated for the levels of 158P3D2 in a given sample (e.g. the levels of circulating 158P3D2 antigen and/or 158P3D2 expressing cells) in order to assist in the determination of the most effective dosing regimen, etc. Such evaluations are also used for monitoring purposes throughout therapy, and are useful to gauge therapeutic success in combination with the evaluation of other parameters (for example, urine cytology and/or ImmunoCyt levels in bladder cancer therapy, or by analogy, serum PSA levels in prostate cancer therapy).

Anti-idiotypic anti-158P3D2 antibodies can also be used in anti-cancer therapy as a vaccine for inducing an immune response to cells expressing a 158P3D2-related protein. In particular, the generation of anti-idiotypic antibodies is well known in the art; this methodology can readily be adapted to generate anti-idiotypic anti-158P3D2 antibodies that mimic an epitope on a 158P3D2-related protein (see, for example, Wagner et al., 1997, Hybridoma 16: 33-40; Foon et al., 1995, J. Clin. Invest. 96:334-342; Herlyn et al., 1996, Cancer Immunol. Immunother. 43:65-76). Such an anti-idiotypic antibody can be used in cancer vaccine strategies.

X.C.) 158P3D2 as a Target for Cellular Immune Responses

Vaccines and methods of preparing vaccines that contain an immunogenically effective amount of one or more HLA-binding peptides as described herein are further embodiments of the invention. Furthermore, vaccines in accordance with the invention encompass compositions of one or more of the claimed peptides. A peptide can be present in a vaccine individually. Alternatively, the peptide can exist as a homopolymer comprising multiple copies of the same peptide, or as a heteropolymer of various peptides. Polymers have the advantage of increased immunological reaction and, where different peptide epitopes are used to make up the polymer, the additional ability to induce antibodies and/or CTLs that react with different antigenic determinants of the pathogenic organism or tumor-related peptide targeted for an immune response. The composition can be a naturally occurring region of an antigen or can be prepared, e.g., recombinantly or by chemical synthesis.

Carriers that can be used with vaccines of the invention are well known in the art, and include, e.g., thyroglobulin, albumins such as human serum albumin, tetanus toxoid, polyamino acids such as poly L-lysine, poly L-glutamic acid, influenza, hepatitis B virus core protein, and the like. The vaccines can contain a physiologically tolerable (i.e., acceptable) diluent such as water, or saline, preferably phosphate buffered saline. The vaccines also typically include an adjuvant. Adjuvants such as incomplete Freund's adjuvant, aluminum phosphate, aluminum hydroxide, or alum are examples of materials well known in the art. Additionally, as disclosed herein, CTL responses can be primed by conjugating peptides of the invention to lipids, such as tripalmitoyl-S-glycerylcysteinlyseryl-serine (P₃CSS). Moreover, an adjuvant such as a synthetic cytosine-phosphorothiolated-guanine-containing (CpG) oligonucleotides has been found to increase CTL responses 10- to 100-fold. (see, e.g. Davila and Celis J. Immunol. 165:539-547 (2000))

Upon immunization with a peptide composition in accordance with the invention, via injection, aerosol, oral, transdermal, transmucosal, intrapleural, intrathecal, or other suitable routes, the immune system of the host responds to the vaccine by producing large amounts of CTLs and/or HTLs specific for the desired antigen. Consequently, the host becomes at least partially immune to later development of cells that express or overexpress 158P3D2 antigen, or derives at least some therapeutic benefit when the antigen was tumor-associated.

In some embodiments, it may be desirable to combine the class I peptide components with components that induce or facilitate neutralizing antibody and or helper T cell responses directed to the target antigen. A preferred embodiment of such a composition comprises class I and class II epitopes in accordance with the invention. An alternative embodiment of such a composition comprises a class I and/or class II epitope in accordance with the invention, along with a cross reactive HTL epitope such as PADRE™ (Epimmune, San Diego, Calif.) molecule (described e.g., in U.S. Pat. No. 5,736,142).

A vaccine of the invention can also include antigen-presenting cells (APC), such as dendritic cells (DC), as a vehicle to present peptides of the invention. Vaccine compositions can be created in vitro, following dendritic cell mobilization and harvesting, whereby loading of dendritic cells occurs in vitro. For example, dendritic cells are transfected, e.g., with a minigene in accordance with the invention, or are pulsed with peptides. The dendritic cell can then be administered to a patient to elicit immune responses in vivo. Vaccine compositions, either DNA- or peptide-based, can also be administered in vivo in combination with dendritic cell mobilization whereby loading of dendritic cells occurs in vivo.

Preferably, the following principles are utilized when selecting an array of epitopes for inclusion in a polyepitopic composition for use in a vaccine, or for selecting discrete epitopes to be included in a vaccine and/or to be encoded by nucleic acids such as a minigene. It is preferred that each of the following principles be balanced in order to make the selection. The multiple epitopes to be incorporated in a given vaccine composition may be, but need not be, contiguous in sequence in the native antigen from which the epitopes are derived.

1.) Epitopes are selected which, upon administration, mimic immune responses that have been observed to be correlated with tumor clearance. For HLA Class I this includes 3-4 epitopes that come from at least one tumor associated antigen (TAA). For HLA Class II a similar rationale is employed; again 3-4 epitopes are selected from at least one TAA (see, e.g., Rosenberg et al., Science 278:1447-1450). Epitopes from one TAA may be used in combination with epitopes from one or more additional TAAs to produce a vaccine that targets tumors with varying expression patterns of frequently-expressed TAAs.

2.) Epitopes are selected that have the requisite binding affinity established to be correlated with immunogenicity: for HLA Class I an IC₅₀ of 500 nM or less, often 200 nM or less; and for Class II an IC₅₀ of 1000 nM or less.

3.) Sufficient supermotif bearing-peptides, or a sufficient array of allele-specific motif-bearing peptides, are selected to give broad population coverage. For example, it is preferable to have at least 80% population coverage. A Monte Carlo analysis, a statistical evaluation known in the art, can be employed to assess the breadth, or redundancy of, population coverage.

4.) When selecting epitopes from cancer-related antigens it is often useful to select analogs because the patient may have developed tolerance to the native epitope.

5.) Of particular relevance are epitopes referred to as “nested epitopes.” Nested epitopes occur where at least two epitopes overlap in a given peptide sequence. A nested peptide sequence can comprise B cell, HLA class I and/or HLA class II epitopes. When providing nested epitopes, a general objective is to provide the greatest number of epitopes per sequence. Thus, an aspect is to avoid providing a peptide that is any longer than the amino terminus of the amino terminal epitope and the carboxyl terminus of the carboxyl terminal epitope in the peptide. When providing a multi-epitopic sequence, such as a sequence comprising nested epitopes, it is generally important to screen the sequence in order to insure that it does not have pathological or other deleterious biological properties.

6.) If a polyepitopic protein is created, or when creating a minigene, an objective is to generate the smallest peptide that encompasses the epitopes of interest. This principle is similar, if not the same as that employed when selecting a peptide comprising nested epitopes. However, with an artificial polyepitopic peptide, the size minimization objective is balanced against the need to integrate any spacer sequences between epitopes in the polyepitopic protein. Spacer amino acid residues can, for example, be introduced to avoid junctional epitopes (an epitope recognized by the immune system, not present in the target antigen, and only created by the man-made juxtaposition of epitopes), or to facilitate cleavage between epitopes and thereby enhance epitope presentation. Junctional epitopes are generally to be avoided because the recipient may generate an immune response to that non-native epitope. Of particular concern is a junctional epitope that is a “dominant epitope.” A dominant epitope may lead to such a zealous response that immune responses to other epitopes are diminished or suppressed.

7.) Where the sequences of multiple variants of the same target protein are present, potential peptide epitopes can also be selected on the basis of their conservancy. For example, a criterion for conservancy may define that the entire sequence of an HLA class I binding peptide or the entire 9-mer core of a class II binding peptide be conserved in a designated percentage of the sequences evaluated for a specific protein antigen.

X.C.1.) Minigene Vaccines

A number of different approaches are available which allow simultaneous delivery of multiple epitopes. Nucleic acids encoding the peptides of the invention are a particularly useful embodiment of the invention. Epitopes for inclusion in a minigene are preferably selected according to the guidelines set forth in the previous section. A preferred means of administering nucleic acids encoding the peptides of the invention uses minigene constructs encoding a peptide comprising one or multiple epitopes of the invention.

The use of multi-epitope minigenes is described below and in, Ishioka et al., J. Immunol. 162:3915-3925, 1999; An, L. and Whitton, J. L., J. Virol. 71:2292, 1997; Thomson, S. A. et al., J. Immunol. 157:822, 1996; Whitton, J. L. et al., J. Virol. 67:348, 1993; Hanke, R. et al., Vaccine 16:426, 1998. For example, a multi-epitope DNA plasmid encoding supermotif- and/or motif-bearing epitopes derived 158P3D2, the PADRE® universal helper T cell epitope (or multiple HTL epitopes from 158P3D2), and an endoplasmic reticulum-translocating signal sequence can be engineered. A vaccine may also comprise epitopes that are derived from other TAAs.

The immunogenicity of a multi-epitopic minigene can be confirmed in transgenic mice to evaluate the magnitude of CTL induction responses against the epitopes tested. Further, the immunogenicity of DNA-encoded epitopes in vivo can be correlated with the in vitro responses of specific CTL lines against target cells transfected with the DNA plasmid. Thus, these experiments can show that the minigene serves to both: 1.) generate a CTL response and 2.) that the induced CTLs recognized cells expressing the encoded epitopes.

For example, to create a DNA sequence encoding the selected epitopes (minigene) for expression in human cells, the amino acid sequences of the epitopes may be reverse translated. A human codon usage table can be used to guide the codon choice for each amino acid. These epitope-encoding DNA sequences may be directly adjoined, so that when translated, a continuous polypeptide sequence is created. To optimize expression and/or immunogenicity, additional elements can be incorporated into the minigene design. Examples of amino acid sequences that can be reverse translated and included in the minigene sequence include: HLA class I epitopes, HLA class II epitopes, antibody epitopes, a ubiquitination signal sequence, and/or an endoplasmic reticulum targeting signal. In addition, HLA presentation of CTL and HTL epitopes may be improved by including synthetic (e.g. poly-alanine) or naturally-occurring flanking sequences adjacent to the CTL or HTL epitopes; these larger peptides comprising the epitope(s) are within the scope of the invention.

The minigene sequence may be converted to DNA by assembling oligonucleotides that encode the plus and minus strands of the minigene. Overlapping oligonucleotides (30-100 bases long) may be synthesized, phosphorylated, purified and annealed under appropriate conditions using well known techniques. The ends of the oligonucleotides can be joined, for example, using T4 DNA ligase. This synthetic minigene, encoding the epitope polypeptide, can then be cloned into a desired expression vector.

Standard regulatory sequences well known to those of skill in the art are preferably included in the vector to ensure expression in the target cells. Several vector elements are desirable: a promoter with a down-stream cloning site for minigene insertion; a polyadenylation signal for efficient transcription termination; an E. coli origin of replication; and an E. coli selectable marker (e.g. ampicillin or kanamycin resistance). Numerous promoters can be used for this purpose, e.g., the human cytomegalovirus (hCMV) promoter. See, e.g., U.S. Pat. Nos. 5,580,859 and 5,589,466 for other suitable promoter sequences.

Additional vector modifications may be desired to optimize minigene expression and immunogenicity. In some cases, introns are required for efficient gene expression, and one or more synthetic or naturally-occurring introns could be incorporated into the transcribed region of the minigene. The inclusion of mRNA stabilization sequences and sequences for replication in mammalian cells may also be considered for increasing minigene expression.

Once an expression vector is selected, the minigene is cloned into the polylinker region downstream of the promoter. This plasmid is transformed into an appropriate E. coli strain, and DNA is prepared using standard techniques. The orientation and DNA sequence of the minigene, as well as all other elements included in the vector, are confirmed using restriction mapping and DNA sequence analysis. Bacterial cells harboring the correct plasmid can be stored as a master cell bank and a working cell bank.

In addition, immunostimulatory sequences (ISSs or CpGs) appear to play a role in the immunogenicity of DNA vaccines. These sequences may be included in the vector, outside the minigene coding sequence, if desired to enhance immunogenicity.

In some embodiments, a bi-cistronic expression vector which allows production of both the minigene-encoded epitopes and a second protein (included to enhance or decrease immunogenicity) can be used. Examples of proteins or polypeptides that could beneficially enhance the immune response if co-expressed include cytokines (e.g., IL-2, IL-12, GM-CSF), cytokine-inducing molecules (e.g., LeIF), costimulatory molecules, or for HTL responses, pan-DR binding proteins (PADRE™, Epimmune, San Diego, Calif.). Helper (HTL) epitopes can be joined to intracellular targeting signals and expressed separately from expressed CTL epitopes; this allows direction of the HTL epitopes to a cell compartment different than that of the CTL epitopes. If required, this could facilitate more efficient entry of HTL epitopes into the HLA class II pathway, thereby improving HTL induction. In contrast to HTL or CTL induction, specifically decreasing the immune response by co-expression of immunosuppressive molecules (e.g. TGF-β) may be beneficial in certain diseases.

Therapeutic quantities of plasmid DNA can be produced for example, by fermentation in E. coli, followed by purification. Aliquots from the working cell bank are used to inoculate growth medium, and grown to saturation in shaker flasks or a bioreactor according to well-known techniques. Plasmid DNA can be purified using standard bioseparation technologies such as solid phase anion-exchange resins supplied by QIAGEN, Inc. (Valencia, Calif.). If required, supercoiled DNA can be isolated from the open circular and linear forms using gel electrophoresis or other methods.

Purified plasmid DNA can be prepared for injection using a variety of formulations. The simplest of these is reconstitution of lyophilized DNA in sterile phosphate-buffer saline (PBS). This approach, known as “naked DNA,” is currently being used for intramuscular (IM) administration in clinical trials. To maximize the immunotherapeutic effects of minigene DNA vaccines, an alternative method for formulating purified plasmid DNA may be desirable. A variety of methods have been described, and new techniques may become available. Cationic lipids, glycolipids, and fusogenic liposomes can also be used in the formulation (see, e.g., as described by WO 93/24640; Mannino & Gould-Fogerite, BioTechniques 6(7): 682 (1988); U.S. Pat. No. 5,279,833; WO 91/06309; and Felgner, et al., Proc. Nat'l Acad. Sci. USA 84:7413 (1987). In addition, peptides and compounds referred to collectively as protective, interactive, non-condensing compounds (PINC) could also be complexed to purified plasmid DNA to influence variables such as stability, intramuscular dispersion, or trafficking to specific organs or cell types.

Target cell sensitization can be used as a functional assay for expression and HLA class I presentation of minigene-encoded CTL epitopes. For example, the plasmid DNA is introduced into a mammalian cell line that is suitable as a target for standard CTL chromium release assays. The transfection method used will be dependent on the final formulation. Electroporation can be used for “naked” DNA, whereas cationic lipids allow direct in vitro transfection. A plasmid expressing green fluorescent protein (GFP) can be co-transfected to allow enrichment of transfected cells using fluorescence activated cell sorting (FACS). These cells are then chromium-51 (⁵¹Cr) labeled and used as target cells for epitope-specific CTL lines; cytolysis, detected by ⁵¹Cr release, indicates both production of, and HLA presentation of, minigene-encoded CTL epitopes. Expression of HTL epitopes may be evaluated in an analogous manner using assays to assess HTL activity.

In vivo immunogenicity is a second approach for functional testing of minigene DNA formulations. Transgenic mice expressing appropriate human HLA proteins are immunized with the DNA product. The dose and route of administration are formulation dependent (e.g., IM for DNA in PBS, intraperitoneal (i.p.) for lipid-complexed DNA). Twenty-one days after immunization, splenocytes are harvested and restimulated for one week in the presence of peptides encoding each epitope being tested. Thereafter, for CTL effector cells, assays are conducted for cytolysis of peptide-loaded, ⁵¹Cr-labeled target cells using standard techniques. Lysis of target cells that were sensitized by HLA loaded with peptide epitopes, corresponding to minigene-encoded epitopes, demonstrates DNA vaccine function for in vivo induction of CTLs. Immunogenicity of HTL epitopes is confirmed in transgenic mice in an analogous manner.

Alternatively, the nucleic acids can be administered using ballistic delivery as described, for instance, in U.S. Pat. No. 5,204,253. Using this technique, particles comprised solely of DNA are administered. In a further alternative embodiment, DNA can be adhered to particles, such as gold particles.

Minigenes can also be delivered using other bacterial or viral delivery systems well known in the art, e.g., an expression construct encoding epitopes of the invention can be incorporated into a viral vector such as vaccinia.

X.C.2.) Combinations of CTL Peptides with Helper Peptides

Vaccine compositions comprising CTL peptides of the invention can be modified, e.g., analoged, to provide desired attributes, such as improved serum half life, broadened population coverage or enhanced immunogenicity.

For instance, the ability of a peptide to induce CTL activity can be enhanced by linking the peptide to a sequence which contains at least one epitope that is capable of inducing a T helper cell response. Although a CTL peptide can be directly linked to a T helper peptide, often CTL epitope/HTL epitope conjugates are linked by a spacer molecule. The spacer is typically comprised of relatively small, neutral molecules, such as amino acids or amino acid mimetics, which are substantially uncharged under physiological conditions. The spacers are typically selected from, e.g., Ala, Gly, or other neutral spacers of nonpolar amino acids or neutral polar amino acids. It will be understood that the optionally present spacer need not be comprised of the same residues and thus may be a hetero- or homo-oligomer. When present, the spacer will usually be at least one or two residues, more usually three to six residues and sometimes 10 or more residues. The CTL peptide epitope can be linked to the T helper peptide epitope either directly or via a spacer either at the amino or carboxy terminus of the CTL peptide. The amino terminus of either the immunogenic peptide or the T helper peptide may be acylated.

In certain embodiments, the T helper peptide is one that is recognized by T helper cells present in a majority of a genetically diverse population. This can be accomplished by selecting peptides that bind to many, most, or all of the HLA class II molecules. Examples of such amino acid bind many HLA Class II molecules include sequences from antigens such as tetanus toxoid at positions 830-843 (QYIKANSKFIGITE; SEQ ID NO:_(—)1_), Plasmodium falciparum circumsporozoite (CS) protein at positions 378-398 (DIEKKIAKMEKASSVFNVVNS; SEQ ID NO:_(—)2_), and Streptococcus 18 kD protein at positions 116-131 (GAVDSILGGVATYGAA; SEQ ID NO:_(—)3_). Other examples include peptides bearing a DR 1-4-7 supermotif, or either of the DR3 motifs.

Alternatively, it is possible to prepare synthetic peptides capable of stimulating T helper lymphocytes, in a loosely HLA-restricted fashion, using amino acid sequences not found in nature (see, e.g., PCT publication WO 95/07707). These synthetic compounds called Pan-DR-binding epitopes (e.g., PADRE™, Epimmune, Inc., San Diego, Calif.) are designed to most preferably bind most HLA-DR (human HLA class II) molecules. For instance, a pan-DR-binding epitope peptide having the formula: aKXVAAWTLKAAa (SEQ ID NO:_(—)4_), where “X” is either cyclohexylalanine, phenylalanine, or tyrosine, and a is either D-alanine or L-alanine, has been found to bind to most HLA-DR alleles, and to stimulate the response of T helper lymphocytes from most individuals, regardless of their HLA type. An alternative of a pan-DR binding epitope comprises all “L” natural amino acids and can be provided in the form of nucleic acids that encode the epitope.

HTL peptide epitopes can also be modified to alter their biological properties. For example, they can be modified to include D-amino acids to increase their resistance to proteases and thus extend their serum half life, or they can be conjugated to other molecules such as lipids, proteins, carbohydrates, and the like to increase their biological activity. For example, a T helper peptide can be conjugated to one or more palmitic acid chains at either the amino or carboxyl termini.

X.C.3.) Combinations of CTL Peptides with T Cell Priming Agents

In some embodiments it may be desirable to include in the pharmaceutical compositions of the invention at least one component which primes B lymphocytes or T lymphocytes. Lipids have been identified as agents capable of priming CTL in vivo. For example, palmitic acid residues can be attached to the ε- and α-amino groups of a lysine residue and then linked, e.g., via one or more linking residues such as Gly, Gly-Gly-, Ser, Ser-Ser, or the like, to an immunogenic peptide. The lipidated peptide can then be administered either directly in a micelle or particle, incorporated into a liposome, or emulsified in an adjuvant, e.g., incomplete Freund's adjuvant. In a preferred embodiment, a particularly effective immunogenic composition comprises palmitic acid attached to ε- and α-amino groups of Lys, which is attached via linkage, e.g., Ser-Ser, to the amino terminus of the immunogenic peptide.

As another example of lipid priming of CTL responses, E. coli lipoproteins, such as tripalmitoyl-S-glycerylcysteinlyseryl-serine (P₃CSS) can be used to prime virus specific CTL when covalently attached to an appropriate peptide (see, e.g., Deres, et al., Nature 342:561, 1989). Peptides of the invention can be coupled to P₃CSS, for example, and the lipopeptide administered to an individual to specifically prime an immune response to the target antigen. Moreover, because the induction of neutralizing antibodies can also be primed with P₃CSS-conjugated epitopes, two such compositions can be combined to more effectively elicit both humoral and cell-mediated responses.

X.C.4.) Vaccine Compositions Comprising DC Pulsed with CTL and/or HTL Peptides

An embodiment of a vaccine composition in accordance with the invention comprises ex vivo administration of a cocktail of epitope-bearing peptides to PBMC, or isolated DC therefrom, from the patient's blood. A pharmaceutical to facilitate harvesting of DC can be used, such as Progenipoietin™ (Pharmacia-Monsanto, St. Louis, Mo.) or GM-CSF/IL-4. After pulsing the DC with peptides and prior to reinfusion into patients, the DC are washed to remove unbound peptides. In this embodiment, a vaccine comprises peptide-pulsed DCs which present the pulsed peptide epitopes complexed with HLA molecules on their surfaces.

The DC can be pulsed ex vivo with a cocktail of peptides, some of which stimulate CTL responses to 158P3D2. Optionally, a helper T cell (HTL) peptide, such as a natural or artificial loosely restricted HLA Class II peptide, can be included to facilitate the CTL response. Thus, a vaccine in accordance with the invention is used to treat a cancer which expresses or overexpresses 158P3D2.

X.D.) Adoptive Immunotherapy

Antigenic 158P3D2-related peptides are used to elicit a CTL and/or HTL response ex vivo, as well. The resulting CTL or HTL cells, can be used to treat tumors in patients that do not respond to other conventional forms of therapy, or will not respond to a therapeutic vaccine peptide or nucleic acid in accordance with the invention. Ex vivo CTL or HTL responses to a particular antigen are induced by incubating in tissue culture the patient's, or genetically compatible, CTL or HTL precursor cells together with a source of antigen-presenting cells (APC), such as dendritic cells, and the appropriate immunogenic peptide. After an appropriate incubation time (typically about 7-28 days), in which the precursor cells are activated and expanded into effector cells, the cells are infused back into the patient, where they will destroy (CTL) or facilitate destruction (HTL) of their specific target cell (e.g., a tumor cell). Transfected dendritic cells may also be used as antigen presenting cells.

X.E.) Administration of Vaccines for Therapeutic or Prophylactic Purposes

Pharmaceutical and vaccine compositions of the invention are typically used to treat and/or prevent a cancer that expresses or overexpresses 158P3D2. In therapeutic applications, peptide and/or nucleic acid compositions are administered to a patient in an amount sufficient to elicit an effective B cell, CTL and/or HTL response to the antigen and to cure or at least partially arrest or slow symptoms and/or complications. An amount adequate to accomplish this is defined as “therapeutically effective dose.” Amounts effective for this use will depend on, e.g., the particular composition administered, the manner of administration, the stage and severity of the disease being treated, the weight and general state of health of the patient, and the judgment of the prescribing physician.

For pharmaceutical compositions, the immunogenic peptides of the invention, or DNA encoding them, are generally administered to an individual already bearing a tumor that expresses 158P3D2. The peptides or DNA encoding them can be administered individually or as fusions of one or more peptide sequences. Patients can be treated with the immunogenic peptides separately or in conjunction with other treatments, such as surgery, as appropriate.

For therapeutic use, administration should generally begin at the first diagnosis of 158P3D2-associated cancer. This is followed by boosting doses until at least symptoms are substantially abated and for a period thereafter. The embodiment of the vaccine composition (i.e., including, but not limited to embodiments such as peptide cocktails, polyepitopic polypeptides, minigenes, or TAA-specific CTLs or pulsed dendritic cells) delivered to the patient may vary according to the stage of the disease or the patient's health status. For example, in a patient with a tumor that expresses 158P3D2, a vaccine comprising 158P3D2-specific CTL may be more efficacious in killing tumor cells in patient with advanced disease than alternative embodiments.

It is generally important to provide an amount of the peptide epitope delivered by a mode of administration sufficient to effectively stimulate a cytotoxic T cell response; compositions which stimulate helper T cell responses can also be given in accordance with this embodiment of the invention.

The dosage for an initial therapeutic immunization generally occurs in a unit dosage range where the lower value is about 1, 5, 50, 500, or 1,000 μg and the higher value is about 10,000; 20,000; 30,000; or 50,000 μg. Dosage values for a human typically range from about 500 μg to about 50,000 μg per 70 kilogram patient. Boosting dosages of between about 1.0 μg to about 50,000 μg of peptide pursuant to a boosting regimen over weeks to months may be administered depending upon the patient's response and condition as determined by measuring the specific activity of CTL and HTL obtained from the patient's blood. Administration should continue until at least clinical symptoms or laboratory tests indicate that the neoplasia, has been eliminated or reduced and for a period thereafter. The dosages, routes of administration, and dose schedules are adjusted in accordance with methodologies known in the art.

In certain embodiments, the peptides and compositions of the present invention are employed in serious disease states, that is, life-threatening or potentially life threatening situations. In such cases, as a result of the minimal amounts of extraneous substances and the relative nontoxic nature of the peptides in preferred compositions of the invention, it is possible and may be felt desirable by the treating physician to administer substantial excesses of these peptide compositions relative to these stated dosage amounts.

The vaccine compositions of the invention can also be used purely as prophylactic agents. Generally the dosage for an initial prophylactic immunization generally occurs in a unit dosage range where the lower value is about 1, 5, 50, 500, or 1000 μg and the higher value is about 10,000; 20,000; 30,000; or 50,000 μg. Dosage values for a human typically range from about 500 μg to about 50,000 μg per 70 kilogram patient. This is followed by boosting dosages of between about 1.0 μg to about 50,000 μg of peptide administered at defined intervals from about four weeks to six months after the initial administration of vaccine. The immunogenicity of the vaccine can be assessed by measuring the specific activity of CTL and HTL obtained from a sample of the patient's blood.

The pharmaceutical compositions for therapeutic treatment are intended for parenteral, topical, oral, nasal, intrathecal, or local (e.g. as a cream or topical ointment) administration. Preferably, the pharmaceutical compositions are administered parentally, e.g., intravenously, subcutaneously, intradermally, or intramuscularly. Thus, the invention provides compositions for parenteral administration which comprise a solution of the immunogenic peptides dissolved or suspended in an acceptable carrier, preferably an aqueous carrier.

A variety of aqueous carriers may be used, e.g., water, buffered water, 0.8% saline, 0.3% glycine, hyaluronic acid and the like. These compositions may be sterilized by conventional, well-known sterilization techniques, or may be sterile filtered. The resulting aqueous solutions may be packaged for use as is, or lyophilized, the lyophilized preparation being combined with a sterile solution prior to administration.

The compositions may contain pharmaceutically acceptable auxiliary substances as required to approximate physiological conditions, such as pH-adjusting and buffering agents, tonicity adjusting agents, wetting agents, preservatives, and the like, for example, sodium acetate, sodium lactate, sodium chloride, potassium chloride, calcium chloride, sorbitan monolaurate, triethanolamine oleate, etc.

The concentration of peptides of the invention in the pharmaceutical formulations can vary widely, i.e., from less than about 0.1%, usually at or at least about 2% to as much as 20% to 50% or more by weight, and will be selected primarily by fluid volumes, viscosities, etc., in accordance with the particular mode of administration selected.

A human unit dose form of a composition is typically included in a pharmaceutical composition that comprises a human unit dose of an acceptable carrier, in one embodiment an aqueous carrier, and is administered in a volume/quantity that is known by those of skill in the art to be used for administration of such compositions to humans (see, e.g., REMINGTON'S PHARMACEUTICAL SCIENCES, 17^(th) Edition, A. Gennaro, Editor, Mack Publishing Co., Easton, Pa., 1985). For example a peptide dose for initial immunization can be from about 1 to about 50,000 μg, generally 100-5,000 μg, for a 70 kg patient. For example, for nucleic acids an initial immunization may be performed using an expression vector in the form of naked nucleic acid administered IM (or SC or ID) in the amounts of 0.5-5 mg at multiple sites. The nucleic acid (0.1 to 1000 μg) can also be administered using a gene gun. Following an incubation period of 3-4 weeks, a booster dose is then administered. The booster can be recombinant fowlpox virus administered at a dose of 5-10⁷ to 5×10⁹ pfu.

For antibodies, a treatment generally involves repeated administration of the anti-158P3D2 antibody preparation, via an acceptable route of administration such as intravenous injection (IV), typically at a dose in the range of about 0.1 to about 10 mg/kg body weight. In general, doses in the range of 10-500 mg mAb per week are effective and well tolerated. Moreover, an initial loading dose of approximately 4 mg/kg patient body weight IV, followed by weekly doses of about 2 mg/kg IV of the anti-158P3D2 mAb preparation represents an acceptable dosing regimen. As appreciated by those of skill in the art, various factors can influence the ideal dose in a particular case. Such factors include, for example, half life of a composition, the binding affinity of an Ab, the immunogenicity of a substance, the degree of 158P3D2 expression in the patient, the extent of circulating shed 158P3D2 antigen, the desired steady-state concentration level, frequency of treatment, and the influence of chemotherapeutic or other agents used in combination with the treatment method of the invention, as well as the health status of a particular patient. Non-limiting preferred human unit doses are, for example, 500 μg-1 mg, 1 mg-50 mg, 50 mg-100 mg, 100 mg-200 mg, 200 mg-300 mg, 400 mg-500 mg, 500 mg-600 mg, 600 mg-700 mg, 700 mg-800 mg, 800 mg-900 mg, 900 mg-1 g, or 1 mg-700 mg. In certain embodiments, the dose is in a range of 2-5 mg/kg body weight, e.g., with follow on weekly doses of 1-3 mg/kg; 0.5 mg, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10 mg/kg body weight followed, e.g., in two, three or four weeks by weekly doses; 0.5-10 mg/kg body weight, e.g., followed in two, three or four weeks by weekly doses; 225, 250, 275, 300, 325, 350, 375, 400 mg m² of body area weekly; 1-600 mg m² of body area weekly; 225-400 mg m² of body area weekly; these does can be followed by weekly doses for 2, 3, 4, 5, 6, 7, 8, 9, 19, 11, 12 or more weeks.

In one embodiment, human unit dose forms of polynucleotides comprise a suitable dosage range or effective amount that provides any therapeutic effect. As appreciated by one of ordinary skill in the art a therapeutic effect depends on a number of factors, including the sequence of the polynucleotide, molecular weight of the polynucleotide and route of administration. Dosages are generally selected by the physician or other health care professional in accordance with a variety of parameters known in the art, such as severity of symptoms, history of the patient and the like. Generally, for a polynucleotide of about 20 bases, a dosage range may be selected from, for example, an independently selected lower limit such as about 0.1, 0.25, 0.5, 1, 2, 5, 10, 20, 30, 40, 50, 60, 70, 80, 90, 100, 200, 300, 400 or 500 mg/kg up to an independently selected upper limit, greater than the lower limit, of about 60, 80, 100, 200, 300, 400, 500, 750, 1000, 1500, 2000, 3000, 4000, 5000, 6000, 7000, 8000, 9000 or 10,000 mg/kg. For example, a dose may be about any of the following: 0.1 to 100 mg/kg, 0.1 to 50 mg/kg, 0.1 to 25 mg/kg, 0.1 to 10 mg/kg, 1 to 500 mg/kg, 100 to 400 mg/kg, 200 to 300 mg/kg, 1 to 100 mg/kg, 100 to 200 mg/kg, 300 to 400 mg/kg, 400 to 500 mg/kg, 500 to 1000 mg/kg, 500 to 5000 mg/kg, or 500 to 10,000 mg/kg. Generally, parenteral routes of administration may require higher doses of polynucleotide compared to more direct application to the nucleotide to diseased tissue, as do polynucleotides of increasing length.

In one embodiment, human unit dose forms of T-cells comprise a suitable dosage range or effective amount that provides any therapeutic effect. As appreciated by one of ordinary skill in the art, a therapeutic effect depends on a number of factors. Dosages are generally selected by the physician or other health care professional in accordance with a variety of parameters known in the art, such as severity of symptoms, history of the patient and the like. A dose may be about 10⁴ cells to about 10⁶ cells, about 10⁶ cells to about 10⁸ cells, about 10⁸ to about 10¹¹ cells, or about 10⁸ to about 5×10¹⁰ cells. A dose may also about 10⁶ cells/m² to about 10¹⁰ cells/m², or about 10⁶ cells/m² to about 10⁸ cells/m².

Proteins(s) of the invention, and/or nucleic acids encoding the protein(s), can also be administered via liposomes, which may also serve to: 1) target the proteins(s) to a particular tissue, such as lymphoid tissue; 2) to target selectively to diseases cells; or, 3) to increase the half-life of the peptide composition. Liposomes include emulsions, foams, micelles, insoluble monolayers, liquid crystals, phospholipid dispersions, lamellar layers and the like. In these preparations, the peptide to be delivered is incorporated as part of a liposome, alone or in conjunction with a molecule which binds to a receptor prevalent among lymphoid cells, such as monoclonal antibodies which bind to the CD45 antigen, or with other therapeutic or immunogenic compositions. Thus, liposomes either filled or decorated with a desired peptide of the invention can be directed to the site of lymphoid cells, where the liposomes then deliver the peptide compositions. Liposomes for use in accordance with the invention are formed from standard vesicle-forming lipids, which generally include neutral and negatively charged phospholipids and a sterol, such as cholesterol. The selection of lipids is generally guided by consideration of, e.g., liposome size, acid lability and stability of the liposomes in the blood stream. A variety of methods are available for preparing liposomes, as described in, e.g., Szoka, et al., Ann. Rev. Biophys. Bioeng. 9:467 (1980), and U.S. Pat. Nos. 4,235,871, 4,501,728, 4,837,028, and 5,019,369.

For targeting cells of the immune system, a ligand to be incorporated into the liposome can include, e.g., antibodies or fragments thereof specific for cell surface determinants of the desired immune system cells. A liposome suspension containing a peptide may be administered intravenously, locally, topically, etc. in a dose which varies according to, inter alia, the manner of administration, the peptide being delivered, and the stage of the disease being treated.

For solid compositions, conventional nontoxic solid carriers may be used which include, for example, pharmaceutical grades of mannitol, lactose, starch, magnesium stearate, sodium saccharin, talcum, cellulose, glucose, sucrose, magnesium carbonate, and the like. For oral administration, a pharmaceutically acceptable nontoxic composition is formed by incorporating any of the normally employed excipients, such as those carriers previously listed, and generally 10-95% of active ingredient, that is, one or more peptides of the invention, and more preferably at a concentration of 25%-75%.

For aerosol administration, immunogenic peptides are preferably supplied in finely divided form along with a surfactant and propellant. Typical percentages of peptides are about 0.01%-20% by weight, preferably about 1%-10%. The surfactant must, of course, be nontoxic, and preferably soluble in the propellant. Representative of such agents are the esters or partial esters of fatty acids containing from about 6 to 22 carbon atoms, such as caproic, octanoic, lauric, palmitic, stearic, linoleic, linolenic, olesteric and oleic acids with an aliphatic polyhydric alcohol or its cyclic anhydride. Mixed esters, such as mixed or natural glycerides may be employed. The surfactant may constitute about 0.1%-20% by weight of the composition, preferably about 0.25-5%. The balance of the composition is ordinarily propellant. A carrier can also be included, as desired, as with, e.g., lecithin for intranasal delivery.

XI.) DIAGNOSTIC AND PROGNOSTIC EMBODIMENTS OF 158P3D2

As disclosed herein, 158P3D2 polynucleotides, polypeptides, reactive cytotoxic T cells (CTL), reactive helper T cells (HTL) and anti-polypeptide antibodies are used in well known diagnostic, prognostic and therapeutic assays that examine conditions associated with dysregulated cell growth such as cancer, in particular the cancers listed in Table I (see, e.g., both its specific pattern of tissue expression as well as its overexpression in certain cancers as described for example in Example 4). 158P3D2 can be analogized to a prostate associated antigen PSA, the archetypal marker that has been used by medical practitioners for years to identify and monitor the presence of prostate cancer (see, e.g., Merrill et al., J. Urol. 163(2): 503-5120 (2000); Polascik et al., J. Urol. August; 162(2):293-306 (1999) and Fortier et al., J. Nat. Cancer Inst. 91(19): 1635-1640 (1999)). A variety of other diagnostic markers are also used in similar contexts including p53 and K-ras (see, e.g., Tulchinsky et al., Int J Mol Med 1999 Jul. 4(1):99-102 and Minimoto et al., Cancer Detect Prev 2000; 24(1):1-12). Therefore, this disclosure of 158P3D2 polynucleotides and polypeptides (as well as 158P3D2 polynucleotide probes and anti-158P3D2 antibodies used to identify the presence of these molecules) and their properties allows skilled artisans to utilize these molecules in methods that are analogous to those used, for example, in a variety of diagnostic assays directed to examining conditions associated with cancer.

Typical embodiments of diagnostic methods which utilize the 158P3D2 polynucleotides, polypeptides, reactive T cells and antibodies are analogous to those methods from well-established diagnostic assays which employ, e.g., PSA polynucleotides, polypeptides, reactive T cells and antibodies. For example, just as PSA polynucleotides are used as probes (for example in Northern analysis, see, e.g., Sharief et al., Biochem. Mol. Biol. Int. 33(3):567-74 (1994)) and primers (for example in PCR analysis, see, e.g., Okegawa et al., J. Urol. 163(4): 1189-1190 (2000)) to observe the presence and/or the level of PSA mRNAs in methods of monitoring PSA overexpression or the metastasis of prostate cancers, the 158P3D2 polynucleotides described herein can be utilized in the same way to detect 158P3D2 overexpression or the metastasis of prostate and other cancers expressing this gene. Alternatively, just as PSA polypeptides are used to generate antibodies specific for PSA which can then be used to observe the presence and/or the level of PSA proteins in methods to monitor PSA protein overexpression (see, e.g., Stephan et al., Urology 55(4):560-3 (2000)) or the metastasis of prostate cells (see, e.g., Alanen et al., Pathol. Res. Pract. 192(3):233-7 (1996)), the 158P3D2 polypeptides described herein can be utilized to generate antibodies for use in detecting 158P3D2 overexpression or the metastasis of prostate cells and cells of other cancers expressing this gene.

Specifically, because metastases involves the movement of cancer cells from an organ of origin (such as the lung or prostate gland etc.) to a different area of the body (such as a lymph node), assays which examine a biological sample for the presence of cells expressing 158P3D2 polynucleotides and/or polypeptides can be used to provide evidence of metastasis. For example, when a biological sample from tissue that does not normally contain 158P3D2-expressing cells (lymph node) is found to contain 158P3D2-expressing cells such as the 158P3D2 expression seen in LAPC4 and LAPC9, xenografts isolated from lymph node and bone metastasis, respectively, this finding is indicative of metastasis.

Alternatively 158P3D2 polynucleotides and/or polypeptides can be used to provide evidence of cancer, for example, when cells in a biological sample that do not normally express 158P3D2 or express 158P3D2 at a different level are found to express 158P3D2 or have an increased expression of 158P3D2 (see, e.g., the 158P3D2 expression in the cancers listed in Table I and in patient samples etc. shown in the accompanying Figures). In such assays, artisans may further wish to generate supplementary evidence of metastasis by testing the biological sample for the presence of a second tissue restricted marker (in addition to 158P3D2) such as PSA, PSCA etc. (see, e.g., Alanen et al., Pathol. Res. Pract. 192(3): 233-237 (1996)).

Just as PSA polynucleotide fragments and polynucleotide variants are employed by skilled artisans for use in methods of monitoring PSA, 158P3D2 polynucleotide fragments and polynucleotide variants are used in an analogous manner. In particular, typical PSA polynucleotides used in methods of monitoring PSA are probes or primers which consist of fragments of the PSA cDNA sequence. Illustrating this, primers used to PCR amplify a PSA polynucleotide must include less than the whole PSA sequence to function in the polymerase chain reaction. In the context of such PCR reactions, skilled artisans generally create a variety of different polynucleotide fragments that can be used as primers in order to amplify different portions of a polynucleotide of interest or to optimize amplification reactions (see, e.g., Caetano-Anolles, G. Biotechniques 25(3): 472-476, 478-480 (1998); Robertson et al., Methods Mol. Biol. 98:121-154 (1998)). An additional illustration of the use of such fragments is provided in Example 4, where a 158P3D2 polynucleotide fragment is used as a probe to show the expression of 158P3D2 RNAs in cancer cells. In addition, variant polynucleotide sequences are typically used as primers and probes for the corresponding mRNAs in PCR and Northern analyses (see, e.g., Sawai et al., Fetal Diagn. Ther. 1996 Nov.-Dec. 11(6):407-13 and Current Protocols In Molecular Biology, Volume 2, Unit 2, Frederick M. Ausubel et al. eds., 1995)). Polynucleotide fragments and variants are useful in this context where they are capable of binding to a target polynucleotide sequence (e.g., a 158P3D2 polynucleotide shown in FIG. 2 or variant thereof) under conditions of high stringency.

Furthermore, PSA polypeptides which contain an epitope that can be recognized by an antibody or T cell that specifically binds to that epitope are used in methods of monitoring PSA. 158P3D2 polypeptide fragments and polypeptide analogs or variants can also be used in an analogous manner. This practice of using polypeptide fragments or polypeptide variants to generate antibodies (such as anti-PSA antibodies or T cells) is typical in the art with a wide variety of systems such as fusion proteins being used by practitioners (see, e.g., Current Protocols In Molecular Biology, Volume 2, Unit 16, Frederick M. Ausubel et al. eds., 1995). In this context, each epitope(s) functions to provide the architecture with which an antibody or T cell is reactive. Typically, skilled artisans create a variety of different polypeptide fragments that can be used in order to generate immune responses specific for different portions of a polypeptide of interest (see, e.g., U.S. Pat. No. 5,840,501 and U.S. Pat. No. 5,939,533). For example it may be preferable to utilize a polypeptide comprising one of the 158P3D2 biological motifs discussed herein or a motif-bearing subsequence which is readily identified by one of skill in the art based on motifs available in the art. Polypeptide fragments, variants or analogs are typically useful in this context as long as they comprise an epitope capable of generating an antibody or T cell specific for a target polypeptide sequence (e.g. a 158P3D2 polypeptide shown in FIG. 3).

As shown herein, the 158P3D2 polynucleotides and polypeptides (as well as the 158P3D2 polynucleotide probes and anti-158P3D2 antibodies or T cells used to identify the presence of these molecules) exhibit specific properties that make them useful in diagnosing cancers such as those listed in Table I. Diagnostic assays that measure the presence of 158P3D2 gene products, in order to evaluate the presence or onset of a disease condition described herein, such as prostate cancer, are used to identify patients for preventive measures or further monitoring, as has been done so successfully with PSA. Moreover, these materials satisfy a need in the art for molecules having similar or complementary characteristics to PSA in situations where, for example, a definite diagnosis of metastasis of prostatic origin cannot be made on the basis of a test for PSA alone (see, e.g., Alanen et al., Pathol. Res. Pract. 192(3): 233-237 (1996)), and consequently, materials such as 158P3D2 polynucleotides and polypeptides (as well as the 158P3D2 polynucleotide probes and anti-158P3D2 antibodies used to identify the presence of these molecules) need to be employed to confirm a metastases of prostatic origin.

Finally, in addition to their use in diagnostic assays, the 158P3D2 polynucleotides disclosed herein have a number of other utilities such as their use in the identification of oncogenetic associated chromosomal abnormalities in the chromosomal region to which the 158P3D2 gene maps (see Example 3 below). Moreover, in addition to their use in diagnostic assays, the 158P3D2-related proteins and polynucleotides disclosed herein have other utilities such as their use in the forensic analysis of tissues of unknown origin (see, e.g., Takahama K Forensic Sci Int 1996 Jun. 28; 80(1-2): 63-9).

Additionally, 158P3D2-related proteins or polynucleotides of the invention can be used to treat a pathologic condition characterized by the over-expression of 158P3D2. For example, the amino acid or nucleic acid sequence of FIG. 2 or FIG. 3, or fragments of either, can be used to generate an immune response to a 158P3D2 antigen. Antibodies or other molecules that react with 158P3D2 can be used to modulate the function of this molecule, and thereby provide a therapeutic benefit.

XII.) INHIBITION OF 158P3D2 PROTEIN FUNCTION

The invention includes various methods and compositions for inhibiting the binding of 158P3D2 to its binding partner or its association with other protein(s) as well as methods for inhibiting 158P3D2 function.

XII.A.) Inhibition of 158P3D2 with Intracellular Antibodies

In one approach, a recombinant vector that encodes single chain antibodies that specifically bind to 158P3D2 are introduced into 158P3D2 expressing cells via gene transfer technologies. Accordingly, the encoded single chain anti-158P3D2 antibody is expressed intracellularly, binds to 158P3D2 protein, and thereby inhibits its function. Methods for engineering such intracellular single chain antibodies are well known. Such intracellular antibodies, also known as “intrabodies”, are specifically targeted to a particular compartment within the cell, providing control over where the inhibitory activity of the treatment is focused. This technology has been successfully applied in the art (for review, see Richardson and Marasco, 1995, TIBTECH vol. 13). Intrabodies have been shown to virtually eliminate the expression of otherwise abundant cell surface receptors (see, e.g., Richardson et al., 1995, Proc. Natl. Acad. Sci. USA 92: 3137-3141; Beerli et al., 1994, J. Biol. Chem. 289: 23931-23936; Deshane et al., 1994, Gene Ther. 1: 332-337).

Single chain antibodies comprise the variable domains of the heavy and light chain joined by a flexible linker polypeptide, and are expressed as a single polypeptide. Optionally, single chain antibodies are expressed as a single chain variable region fragment joined to the light chain constant region. Well-known intracellular trafficking signals are engineered into recombinant polynucleotide vectors encoding such single chain antibodies in order to precisely target the intrabody to the desired intracellular compartment. For example, intrabodies targeted to the endoplasmic reticulum (ER) are engineered to incorporate a leader peptide and, optionally, a C-terminal ER retention signal, such as the KDEL amino acid motif. Intrabodies intended to exert activity in the nucleus are engineered to include a nuclear localization signal. Lipid moieties are joined to intrabodies in order to tether the intrabody to the cytosolic side of the plasma membrane. Intrabodies can also be targeted to exert function in the cytosol. For example, cytosolic intrabodies are used to sequester factors within the cytosol, thereby preventing them from being transported to their natural cellular destination.

In one embodiment, intrabodies are used to capture 158P3D2 in the nucleus, thereby preventing its activity within the nucleus. Nuclear targeting signals are engineered into such 158P3D2 intrabodies in order to achieve the desired targeting. Such 158P3D2 intrabodies are designed to bind specifically to a particular 158P3D2 domain. In another embodiment, cytosolic intrabodies that specifically bind to a 158P3D2 protein are used to prevent 158P3D2 from gaining access to the nucleus, thereby preventing it from exerting any biological activity within the nucleus (e.g., preventing 158P3D2 from forming transcription complexes with other factors).

In order to specifically direct the expression of such intrabodies to particular cells, the transcription of the intrabody is placed under the regulatory control of an appropriate tumor-specific promoter and/or enhancer. In order to target intrabody expression specifically to prostate, for example, the PSA promoter and/or promoter/enhancer can be utilized (See, for example, U.S. Pat. No. 5,919,652 issued 6 Jul. 1999).

XII.B.) Inhibition of 158P3D2 with Recombinant Proteins

In another approach, recombinant molecules bind to 158P3D2 and thereby inhibit 158P3D2 function. For example, these recombinant molecules prevent or inhibit 158P3D2 from accessing/binding to its binding partner(s) or associating with other protein(s). Such recombinant molecules can, for example, contain the reactive part(s) of a 158P3D2 specific antibody molecule. In a particular embodiment, the 158P3D2 binding domain of a 158P3D2 binding partner is engineered into a dimeric fusion protein, whereby the fusion protein comprises two 158P3D2 ligand binding domains linked to the Fc portion of a human IgG, such as human IgG1. Such IgG portion can contain, for example, the C_(H)2 and C_(H)3 domains and the hinge region, but not the C_(H)1 domain. Such dimeric fusion proteins are administered in soluble form to patients suffering from a cancer associated with the expression of 158P3D2, whereby the dimeric fusion protein specifically binds to 158P3D2 and blocks 158P3D2 interaction with a binding partner. Such dimeric fusion proteins are further combined into multimeric proteins using known antibody linking technologies.

XII.C.) Inhibition of 158P3D2 Transcription or Translation

The present invention also comprises various methods and compositions for inhibiting the transcription of the 158P3D2 gene. Similarly, the invention also provides methods and compositions for inhibiting the translation of 158P3D2 mRNA into protein.

In one approach, a method of inhibiting the transcription of the 158P3D2 gene comprises contacting the 158P3D2 gene with a 158P3D2 antisense polynucleotide. In another approach, a method of inhibiting 158P3D2 mRNA translation comprises contacting a 158P3D2 mRNA with an antisense polynucleotide. In another approach, a 158P3D2 specific ribozyme is used to cleave a 158P3D2 message, thereby inhibiting translation. Such antisense and ribozyme based methods can also be directed to the regulatory regions of the 158P3D2 gene, such as 158P3D2 promoter and/or enhancer elements. Similarly, proteins capable of inhibiting a 158P3D2 gene transcription factor are used to inhibit 158P3D2 mRNA transcription. The various polynucleotides and compositions useful in the aforementioned methods have been described above. The use of antisense and ribozyme molecules to inhibit transcription and translation is well known in the art.

Other factors that inhibit the transcription of 158P3D2 by interfering with 158P3D2 transcriptional activation are also useful to treat cancers expressing 158P3D2. Similarly, factors that interfere with 158P3D2 processing are useful to treat cancers that express 158P3D2. Cancer treatment methods utilizing such factors are also within the scope of the invention.

XII.D.) General Considerations for Therapeutic Strategies

Gene transfer and gene therapy technologies can be used to deliver therapeutic polynucleotide molecules to tumor cells synthesizing 158P3D2 (i.e., antisense, ribozyme, polynucleotides encoding intrabodies and other 158P3D2 inhibitory molecules). A number of gene therapy approaches are known in the art. Recombinant vectors encoding 158P3D2 antisense polynucleotides, ribozymes, factors capable of interfering with 158P3D2 transcription, and so forth, can be delivered to target tumor cells using such gene therapy approaches.

The above therapeutic approaches can be combined with any one of a wide variety of surgical, chemotherapy or radiation therapy regimens. The therapeutic approaches of the invention can enable the use of reduced dosages of chemotherapy (or other therapies) and/or less frequent administration, an advantage for all patients and particularly for those that do not tolerate the toxicity of the chemotherapeutic agent well.

The anti-tumor activity of a particular composition (e.g., antisense, ribozyme, intrabody), or a combination of such compositions, can be evaluated using various in vitro and in vivo assay systems. In vitro assays that evaluate therapeutic activity include cell growth assays, soft agar assays and other assays indicative of tumor promoting activity, binding assays capable of determining the extent to which a therapeutic composition will inhibit the binding of 158P3D2 to a binding partner, etc.

In vivo, the effect of a 158P3D2 therapeutic composition can be evaluated in a suitable animal model. For example, xenogenic prostate cancer models can be used, wherein human prostate cancer explants or passaged xenograft tissues are introduced into immune compromised animals, such as nude or SCID mice (Klein et al., 1997, Nature Medicine 3: 402-408). For example, PCT Patent Application WO98/16628 and U.S. Pat. No. 6,107,540 describe various xenograft models of human prostate cancer capable of recapitulating the development of primary tumors, micrometastasis, and the formation of osteoblastic metastases characteristic of late stage disease. Efficacy can be predicted using assays that measure inhibition of tumor formation, tumor regression or metastasis, and the like.

In vivo assays that evaluate the promotion of apoptosis are useful in evaluating therapeutic compositions. In one embodiment, xenografts from tumor bearing mice treated with the therapeutic composition can be examined for the presence of apoptotic foci and compared to untreated control xenograft-bearing mice. The extent to which apoptotic foci are found in the tumors of the treated mice provides an indication of the therapeutic efficacy of the composition.

The therapeutic compositions used in the practice of the foregoing methods can be formulated into pharmaceutical compositions comprising a carrier suitable for the desired delivery method. Suitable carriers include any material that when combined with the therapeutic composition retains the anti-tumor function of the therapeutic composition and is generally non-reactive with the patient's immune system. Examples include, but are not limited to, any of a number of standard pharmaceutical carriers such as sterile phosphate buffered saline solutions, bacteriostatic water, and the like (see, generally, Remington's Pharmaceutical Sciences 16^(th) Edition, A. Osal., Ed., 1980).

Therapeutic formulations can be solubilized and administered via any route capable of delivering the therapeutic composition to the tumor site. Potentially effective routes of administration include, but are not limited to, intravenous, parenteral, intraperitoneal, intramuscular, intratumor, intradermal, intraorgan, orthotopic, and the like. A preferred formulation for intravenous injection comprises the therapeutic composition in a solution of preserved bacteriostatic water, sterile unpreserved water, and/or diluted in polyvinylchloride or polyethylene bags containing 0.9% sterile Sodium Chloride for Injection, USP. Therapeutic protein preparations can be lyophilized and stored as sterile powders, preferably under vacuum, and then reconstituted in bacteriostatic water (containing for example, benzyl alcohol preservative) or in sterile water prior to injection.

Dosages and administration protocols for the treatment of cancers using the foregoing methods will vary with the method and the target cancer, and will generally depend on a number of other factors appreciated in the art.

XIII.) Kits

For use in the diagnostic and therapeutic applications described herein, kits are also within the scope of the invention. Such kits can comprise a carrier, package or container that is compartmentalized to receive one or more containers such as vials, tubes, and the like, each of the container(s) comprising one of the separate elements to be used in the method. For example, the container(s) can comprise a probe that is or can be detectably labeled. Such probe can be an antibody or polynucleotide specific for a 158P3D2-related protein or a 158P3D2 gene or message, respectively. Where the method utilizes nucleic acid hybridization to detect the target nucleic acid, the kit can also have containers containing nucleotide(s) for amplification of the target nucleic acid sequence and/or a container comprising a reporter-means, such as a biotin-binding protein, such as avidin or streptavidin, bound to a reporter molecule, such as an enzymatic, fluorescent, or radioisotope label. The kit can include all or part of the amino acid sequence of FIG. 2 or FIG. 3 or analogs thereof, or a nucleic acid molecules that encodes such amino acid sequences.

The kit of the invention will typically comprise the container described above and one or more other containers comprising materials desirable from a commercial and user standpoint, including buffers, diluents, filters, needles, syringes, and package inserts with instructions for use.

A label can be present on the container to indicate that the composition is used for a specific therapy or non-therapeutic application, and can also indicate directions for either in vivo or in vitro use, such as those described above. Directions and or other information can also be included on an insert which is included with the kit.

EXAMPLES

Various aspects of the invention are further described and illustrated by way of the several examples that follow, none of which are intended to limit the scope of the invention.

Example 1 SSH-Generated Isolation of a cDNA Fragment of the 158P3D2 Gene

To isolate genes that are over-expressed in bladder cancer, Suppression Subtractive Hybridization (SSH) procedure using cDNA derived from bladder cancer tissues, including invasive transitional cell carcinoma. The 158P3D2 SSH cDNA sequence was derived from a bladder cancer pool minus normal bladder cDNA subtraction. Included in the driver were also cDNAs derived from 9 other normal tissues. The 158P3D2 cDNA was identified as highly expressed in the bladder cancer tissue pool, with lower expression seen in a restricted set of normal tissues.

The SSH DNA sequence of 312 bp (FIG. 1) shows identity to the fer-1-like 4 (C. elegans) (FER1L4) mRNA (FIG. 4A). A 158P3D2 cDNA clone 158P3D2-BCP1 of 1994 bp was isolated from bladder cancer cDNA, revealing an ORF of 328 amino acids (FIG. 2 and FIG. 3).

Amino acid sequence analysis of 158P3D2 reveals 100% identity over 328 amino acid region to dJ477O4.1.1, a novel protein similar to otoferlin and dysferlin, isoform I protein (GenBank Accession CAB89410.1|, FIG. 4B).

The 158P3D2 protein has a transmembrane domain of 23 residues between amino acids 292-313 predicted by the SOSUI Signal program (http://sosui.proteome.bio.tuat.acjp/cgi-bin/sosui.cgi?/sosuisignal/sosuisignal_submit.html).

Materials and Methods

Human Tissues:

The patient cancer and normal tissues were purchased from different sources such as the NDR1 (Philadelphia, Pa.). mRNA for some normal tissues were purchased from Clontech, Palo Alto, Calif.

RNA Isolation:

Tissues were homogenized in Trizol reagent (Life Technologies, Gibco BRL) using 10 ml g tissue isolate total RNA. Poly A RNA was purified from total RNA using Qiagen's Oligotex mRNA Mini and Midi kits. Total and mRNA were quantified by spectrophotometric analysis (O.D. 260/280 nm) and analyzed by gel electrophoresis.

Oligonucleotides:

The following HPLC purified oligonucleotides were used.

DPNCDN (cDNA synthesis primer): (SEQ ID NO:_5_) 5′TTTTGATCAAGCTT₃₀3′ Adaptor 1: (SEQ ID NO:_6_) 5′CTAATACGACTCACTATAGGGCTCGAGCGGCCGCCCGGGCAG3′ (SEQ ID NO:_7_) 3′GGCCCGTCCTAG5′ Adaptor 2: (SEQ ID NO:_8_) 5′GTAATACGACTCACTATAGGGCAGCGTGGTCGCGGCCGAG3′ (SEQ ID NO:_9_) 3′CGGCTCCTAG5′ PCR primer 1: (SEQ ID NO:_10_) 5′CTAATACGACTCACTATAGGGC3′ Nested primer (NP)1: (SEQ ID NO:_11_) 5′TCGAGCGGCCGCCCGGGCAGGA3′ Nested primer (NP)2: (SEQ ID NO:_12_) 5′AGCGTGGTCGCGGCCGAGGA3′

Suppression Subtractive Hybridization:

Suppression Subtractive Hybridization (SSH) was used to identify cDNAs corresponding to genes that may be differentially expressed in bladder cancer. The SSH reaction utilized cDNA from bladder cancer and normal tissues.

The gene 158P3D2 sequence was derived from a bladder cancer pool minus normal bladder cDNA subtraction. The SSH DNA sequence (FIG. 1) was identified.

The cDNA derived from of pool of normal bladder tissues was used as the source of the “driver” cDNA, while the cDNA from a pool of bladder cancer tissues was used as the source of the “tester” cDNA. Double stranded cDNAs corresponding to tester and driver cDNAs were synthesized from 2 μg of poly(A)⁺ RNA isolated from the relevant xenograft tissue, as described above, using CLONTECH's PCR-Select cDNA Subtraction Kit and 1 ng of oligonucleotide DPNCDN as primer. First- and second-strand synthesis were carried out as described in the Kit's user manual protocol (CLONTECH Protocol No. PT1117-1, Catalog No. K1804-1). The resulting cDNA was digested with Dpn II for 3 hrs at 37° C. Digested cDNA was extracted with phenol/chloroform (1:1) and ethanol precipitated.

Driver cDNA was generated by combining in a 1:1 ratio Dpn II digested cDNA from the relevant tissue source (see above) with a mix of digested cDNAs derived from the nine normal tissues: stomach, skeletal muscle, lung, brain, liver, kidney, pancreas, small intestine, and heart.

Tester cDNA was generated by diluting 1 μl of Dpn II digested cDNA from the relevant tissue source (see above) (400 ng) in 5 μl of water. The diluted cDNA (2 μl, 160 ng) was then ligated to 2 μl of Adaptor 1 and Adaptor 2 (10 μM), in separate ligation reactions, in a total volume of 10 μl at 16° C. overnight, using 400 u of T4 DNA ligase (CLONTECH). Ligation was terminated with 1 μl of 0.2 M EDTA and heating at 72° C. for 5 min.

The first hybridization was performed by adding 1.5 μl (600 ng) of driver cDNA to each of two tubes containing 1.5 μl (20 ng) Adaptor 1- and Adaptor 2-ligated tester cDNA. In a final volume of 4 μl, the samples were overlaid with mineral oil, denatured in an MJ Research thermal cycler at 98° C. for 1.5 minutes, and then were allowed to hybridize for 8 hrs at 68° C. The two hybridizations were then mixed together with an additional 1 μl of fresh denatured driver cDNA and were allowed to hybridize overnight at 68° C. The second hybridization was then diluted in 200 μl of 20 mM Hepes, pH 8.3, 50 mM NaCl, 0.2 mM EDTA, heated at 70° C. for 7 min. and stored at −20° C.

PCR Amplification, Cloning and Sequencing of Gene Fragments Generated from SSH:

To amplify gene fragments resulting from SSH reactions, two PCR amplifications were performed. In the primary PCR reaction 1 μl of the diluted final hybridization mix was added to 1 μl of PCR primer 1 (10 μM), 0.5 μl dNTP mix (10 μM), 2.5 μl 10× reaction buffer (CLONTECH) and 0.5 μl 50× Advantage cDNA polymerase Mix (CLONTECH) in a final volume of 25 μl. PCR 1 was conducted using the following conditions: 75° C. for 5 min., 94° C. for 25 sec., then 27 cycles of 94° C. for 10 sec, 66° C. for 30 sec, 72° C. for 1.5 min. Five separate primary PCR reactions were performed for each experiment. The products were pooled and diluted 1:10 with water. For the secondary PCR reaction, 1 μl from the pooled and diluted primary PCR reaction was added to the same reaction mix as used for PCR 1, except that primers NP1 and NP2 (10 μM) were used instead of PCR primer 1. PCR 2 was performed using 10-12 cycles of 94° C. for 10 sec, 68° C. for 30 sec, and 72° C. for 1.5 minutes. The PCR products were analyzed using 2% agarose gel electrophoresis.

The PCR products were inserted into pCR2.1 using the T/A vector cloning kit (Invitrogen). Transformed E. coli were subjected to blue/white and ampicillin selection. White colonies were picked and arrayed into 96 well plates and were grown in liquid culture overnight. To identify inserts, PCR amplification was performed on 1 ml of bacterial culture using the conditions of PCR1 and NP1 and NP2 as primers. PCR products were analyzed using 2% agarose gel electrophoresis.

Bacterial clones were stored in 20% glycerol in a 96 well format. Plasmid DNA was prepared, sequenced, and subjected to nucleic acid homology searches of the GenBank, dBest, and NCI-CGAP databases.

First strand cDNAs can be generated from 1 μg of mRNA with oligo (dT) 12-18 priming using the Gibco-BRL Superscript Preamplification system. The manufacturer's protocol was used which included an incubation for 50 min at 42° C. with reverse transcriptase followed by RNAse H treatment at 37° C. for 20 min. After completing the reaction, the volume can be increased to 200 μl with water prior to normalization. First strand cDNAs from 16 different normal human tissues can be obtained from Clontech.

Normalization of the first strand cDNAs from multiple tissues was performed by using the primers 5′atatcgccgcgctcgtcgtcgacaa3′ (SEQ ID NO:_(—)13_) and 5′agccacacgcagctcattgtagaagg 3′ (SEQ ID NO:_(—)14_) to amplify β-actin. First strand cDNA (5 μl) were amplified in a total volume of 50 μl containing 0.4 μM primers, 0.2 μM each dNTPs, 1×PCR buffer (Clontech, 10 mM Tris-HCL, 1.5 mM MgCl₂, 50 mM KCl, pH8.3) and 1× Klentaq DNA polymerase (Clontech). Five μl of the PCR reaction can be removed at 18, 20, and 22 cycles and used for agarose gel electrophoresis. PCR was performed using an MJ Research thermal cycler under the following conditions: Initial denaturation can be at 94° C. for 15 sec, followed by a 18, 20, and 22 cycles of 94° C. for 15, 65° C. for 2 min, 72° C. for 5 sec. A final extension at 72° C. was carried out for 2 min. After agarose gel electrophoresis, the band intensities of the 283 b.p. β-actin bands from multiple tissues were compared by visual inspection. Dilution factors for the first strand cDNAs were calculated to result in equal β-actin band intensities in all tissues after 22 cycles of PCR. Three rounds of normalization can be required to achieve equal band intensities in all tissues after 22 cycles of PCR.

To determine expression levels of the 158P3D2 gene, 5 μl of normalized first strand cDNA were analyzed by PCR using 26, and 30 cycles of amplification. Semi-quantitative expression analysis can be achieved by comparing the PCR products at cycle numbers that give light band intensities. The primers used for RT-PCR were designed using the 158P3D2 SSH sequence and are listed below:

158P3D2.1 (SEQ ID NO: 15) 5′ CATCTATGTGAAGAGCTGGGTGAA 3′ 158P3D2.2 (SEQ ID NO: 16) 5′ AGGTAGTCAAAGCGGAACACAAAG 3′

A typical RT-PCR expression analysis is shown in FIG. 14. RT-PCR expression analysis was performed on first strand cDNAs generated using pools of tissues from multiple samples. The cDNAs were shown to be normalized using beta-actin PCR. Results show strong expression of 158P3D2 in bladder cancer pool, kidney cancer pool and cancer metastasis pool. Expression of 158P3D2 is also detected in colon cancer pool, lung cancer pool, ovary cancer pool, breast cancer pool, pancreas cancer pool and prostate metastases to lymph node, and vital pool 2, but not vital pool 1.

Example 2 Full Length Cloning of 158P3D2

The 158P3D2 SSH cDNA sequence was derived from a bladder cancer pool minus normal bladder cDNA subtraction. The SSH cDNA sequence (FIG. 1) was designated 158P3D2. The full-length cDNA clone 158P3D2 v.1 clone 158P3D2-BCP1 and 158P3D2-BCP2 (FIG. 2) were cloned from bladder cancer pool cDNA.

The SSH DNA sequence of 312 bp (FIG. 1) shows identity to the fer-1-like 4 (C. elegans) (FER1L4) mRNA (FIG. 4A). A 158P3D2 cDNA clone 158P3D2-BCP1 of 1994 bp was isolated from bladder cancer cDNA, revealing an ORF of 328 amino acids (FIG. 2 and FIG. 3).

Amino acid sequence analysis of 158P3D2 reveals 100% identity over 328 amino acid region to dJ477O4.1.1, a novel protein similar to otoferlin and dysferlin, isoform 1 protein (GenBank Accession CAB89410.1|, FIG. 4B).

The 158P3D2 protein has a transmembrane domain of 23 residues between amino acids 292-313 predicted by the SOSUI Signal program (http://sosui.proteome.bio.tuat.acjp/cgi-bin/sosui.cgi?/sosuisignal/sosuisignal_submit.html).

Example 3 Chromosomal Mapping of 158P3D2

Chromosomal localization can implicate genes in disease pathogenesis. Several chromosome mapping approaches are available including fluorescent in situ hybridization (FISH), human/hamster radiation hybrid (RH) panels (Walter et al., 1994; Nature Genetics 7:22; Research Genetics, Huntsville Ala.), human-rodent somatic cell hybrid panels such as is available from the Coriell Institute (Camden, N.J.), and genomic viewers utilizing BLAST homologies to sequenced and mapped genomic clones (NCBI, Bethesda, Md.).

158P3D2 maps to chromosome 8, using 158P3D2 sequence and the NCBI BLAST tool.

Example 4 Expression Analysis of 158P3D2 in Normal Tissues and Patient Specimens

Expression analysis by RT-PCR demonstrated that 158P3D2 is strongly expressed in bladder cancer patient specimens (FIG. 14). First strand cDNA was prepared from vital pool 1 (liver, lung and kidney), vital pool 2 (pancreas, colon and stomach), prostate cancer metastasis to lymph node from 2 different patients, prostate cancer pool, bladder cancer pool, kidney cancer pool, colon cancer pool, lung cancer pool, ovary cancer pool, breast cancer pool, cancer metastasis pool, and pancreas cancer pool. Normalization was performed by PCR using primers to actin and GAPDH. Semi-quantitative PCR, using primers to 158P3D2, was performed at 26 and 30 cycles of amplification. Results show strong expression of 158P3D2 in bladder cancer pool, kidney cancer pool and cancer metastasis pool. Expression of 158P3D2 is also detected in colon cancer pool, lung cancer pool, ovary cancer pool, breast cancer pool, pancreas cancer pool and prostate metastases to lymph node, and vital pool 2, but not vital pool 1.

Northern blot analysis of 158P3D2 in 16 human normal tissues is shown in FIG. 15. An approximately 8 kb transcript is detected exclusively in placenta. Extensive analysis of expression of 158P3D2 in 76 human tissues shows restricted expression of 158P3D2 in placenta and stomach (FIG. 16). Expression of 158P3D2 in patient cancer specimens and human normal tissues is shown in FIG. 17. RNA was extracted from a pool of three bladder cancers, as well as from normal prostate (NP), normal bladder (NB), normal kidney (NK), normal colon (NC), normal lung (NL) and normal breast (NBr). Northern blot with 10 ug of total RNA/lane was probed with 158P3D2 sequence. The results show expression of 158P3D2 in the bladder cancer pool but not in the normal tissues tested. Analysis of individual patient specimens shows strong expression of 158P3D2 in 8 different bladder cancer tissues tested (FIG. 18). Presence of 158P3D2 transcript is also detected in the bladder cancer cell line SCaBER. The expression observed in normal adjacent tissue (isolated from diseased tissues) but not in normal tissue, isolated from healthy donors, may indicate that these tissues are not fully normal and that 158P3D2 may be expressed in early stage tumors.

The restricted expression of 158P3D2 in normal tissues and the expression detected in bladder cancer, prostate cancer pool, kidney cancer pool, colon cancer pool, lung cancer pool, ovary cancer pool, breast cancer pool, pancreas cancer pool and cancer metastases suggest that 158P3D2 is a potential therapeutic target and a diagnostic marker for human cancers.

Example 5 Transcript Variants of 158P3D2

Transcript variants are variants of matured mRNA from the same gene by alternative transcription or alternative splicing. Alternative transcripts are transcripts from the same gene but start transcription at different points. Splice variants are mRNA variants spliced differently from the same transcript. In eukaryotes, when a multi-exon gene is transcribed from genomic DNA, the initial RNA is spliced to produce functional mRNA, which has only exons and is used for translation into an amino acid sequence. Accordingly, a given gene can have zero to many alternative transcripts and each transcript can have zero to many splice variants. Each transcript variant has a unique exon makeup, and can have different coding and/or non-coding (5′ or 3′ end) portions, from the original transcript. Transcript variants can code for similar or different proteins with the same or a similar function or may encode proteins with different functions, and may be expressed in the same tissue at the same time, or at different tissue, or at different times, proteins encoded by transcript variants can have similar or different cellular or extracellular localizations, i.e., be secreted.

Transcript variants are identified by a variety of art-accepted methods. For example, alternative transcripts and splice variants are identified through full-length cloning experiments, or by use of full-length transcript and EST sequences. First, all human ESTs were grouped into clusters which show direct or indirect identity with each other. Second, ESTs in the same cluster were further grouped into sub-clusters and assembled into a consensus sequence. The original gene sequence is compared to the consensus sequence(s) or other full-length sequences. Each consensus sequence is a potential splice variant for that gene. Even when a variant is identified that is not a full-length clone, that portion of the variant is very useful for antigen generation and for further cloning of the full-length splice variant, using techniques known in the art.

Moreover, computer programs are available in the art that identify transcript variants based on genomic sequences. Genomic-based transcript variant identification programs include FgenesH (A. Salamov and V. Solovyev, “Ab initio gene finding in Drosophila genomic DNA,” Genome Research. 2000 April; 10(4):516-22); Grail and GenScan. For a general discussion of splice variant identification protocols see., e.g., Southan, C., A genomic perspective on human proteases, FEBS Lett. 2001 Jun. 8; 498(2-3):214-8; de Souza, S. J., et al., Identification of human chromosome 22 transcribed sequences with ORF expressed sequence tags, Proc. Natl Acad Sci USA. 2000 Nov. 7; 97(23):12690-3.

To further confirm the parameters of a transcript variant, a variety of techniques are available in the art, such as full-length cloning, proteomic validation, PCR-based validation, and 5′ RACE validation, etc. (see e.g., Proteomic Validation: Brennan, S. O., et al., Albumin banks peninsula: a new termination variant characterized by electrospray mass spectrometry, Biochem Biophys Acta. 1999 Aug. 17; 1433(1-2):321-6; Ferranti P, et al., Differential splicing of pre-messenger RNA produces multiple forms of mature caprine alpha(s1)-casein, Eur J. Biochem. 1997 Oct. 1; 249(1):1-7. For PCR-based Validation: Wellmann S, et al., Specific reverse transcription-PCR quantification of vascular endothelial growth factor (VEGF) splice variants by LightCycler technology, Clin Chem. 2001 April; 47(4):654-60; Jia, H. P., et al., Discovery of new human beta-defensins using a genomics-based approach, Gene. 2001 Jan. 24; 263(1-2):211-8. For PCR-based and 5′ RACE Validation: Brigle, K. E., et al., Organization of the murine reduced folate carrier gene and identification of variant splice forms, Biochem Biophys Acta. 1997 Aug. 7; 1353(2): 191-8).

It is known in the art that genomic regions are modulated in cancers. When the genomic region to which a gene maps is modulated in a particular cancer, the alternative transcripts or splice variants of the gene are modulated as well. Disclosed herein is that 158P3D2 has a particular expression profile related to cancer. Alternative transcripts and splice variants of 158P3D2 may also be involved in cancer in the same or different tissues, thus serving as tumor-associated markers/antigens.

The exon composition of the original transcript, designated as 158P3D2 var1, is shown in FIG. 13 and Table XXIIIA. Using the full-length gene and EST sequences, one alternative transcript was identified, designated as 158P3D2 var2, which is also shown in FIG. 13 and Table XXIIIB. Transcript variant 158P3D2 var2 has two potential open reading frames and two protein products, designated as 158P3D2 var2a and 158P3D2 var2b. FIG. 13 shows the schematic alignment of exons of the two transcripts. Potentially, each different combination of exons in spatial order, e.g. exons 1, 2, 3, 4 and 7, can be a splice variant.

Tables XXIV through XXVII are set forth herein on a variant-by-variant basis. Table XXIV shows nucleotide sequence of a transcript variant. Table XXV shows the alignment of the transcript variant 158P3D2 var2 with nucleic acid sequence of 158P3D2 var1. Table XXVI lays out amino acid translation of the transcript variant 158P3D2 var2 for the identified reading frame orientation. Table XXVII displays alignments of the amino acid sequence encoded by the transcript variant 158P3D2 var2 with that of 158P3D2 var1.

Example 6 Single Nucleotide Polymorphisms of 158P3D2

Single Nucleotide Polymorphism (SNP) is a single base pair variation in nucleotide sequences. At a specific point of the genome, there are four possible nucleotide base pairs: A/T, C/G, G/C and T/A. Genotype refers to the base pair make-up of one or more spots in the genome of an individual, while haplotype refers to base pair make-up of more than one varied spots on the same DNA molecule (chromosome in higher organism). SNPs that occur on a cDNA are called cSNPs. These cSNPs may change amino acids of the protein encoded by the gene and thus change the functions of the protein. Some SNPs cause inherited diseases and some others contribute to quantitative variations in phenotype and reactions to environmental factors including diet and drugs among individuals. Therefore, SNPs and/or combinations of alleles (called haplotypes) have many applications including diagnosis of inherited diseases, determination of drug reactions and dosage, identification of genes responsible for diseases and discovery of genetic relationship between individuals (P. Nowotny, J. M. Kwon and A. M. Goate, “SNP analysis to dissect human traits,” Curr. Opin. Neurobiol. 2001 October; 11(5):637-641; M. Pirmohamed and B. K. Park, “Genetic susceptibility to adverse drug reactions,” Trends Pharmacol. Sci. 2001 June; 22(6):298-305; J. H. Riley, C. J. Allan, E. Lai and A. Roses, “The use of single nucleotide polymorphisms in the isolation of common disease genes,” Pharmacogenomics. 2000 February; 1(1):39-47; R. Judson, J. C. Stephens and A. Windemuth, “The predictive power of haplotypes in clinical response,” Pharmacogenomics. 2000 February; 1(1):15-26).

SNPs are identified by a variety of art-accepted methods (P. Bean, “The promising voyage of SNP target discovery,” Am. Clin. Lab. 2001 October-November; 20(9):18-20; K. M. Weiss, “In search of human variation,” Genome Res. 1998 July; 8(7):691-697; M. M. She, “Enabling large-scale pharmacogenetic studies by high-throughput mutation detection and genotyping technologies,” Clin. Chem. 2001 February; 47(2):164-172). For example, SNPs are identified by sequencing DNA fragments that show polymorphism by gel-based methods such as restriction fragment length polymorphism (RFLP) and denaturing gradient gel electrophoresis (DGGE). They can also be discovered by direct sequencing of DNA samples pooled from different individuals or by comparing sequences from different DNA samples. With the rapid accumulation of sequence data in public and private databases, one can discover SNPs by comparing sequences using computer programs (Z. Gu, L. Hillier and P. Y. Kwok, “Single nucleotide polymorphism hunting in cyberspace,” Hum. Mutat. 1998; 12(4):221-225). SNPs can be verified and genotype or haplotype of an individual can be determined by a variety of methods including direct sequencing and high throughput microarrays (P. Y. Kwok, “Methods for genotyping single nucleotide polymorphisms,” Annu. Rev. Genomics Hum. Genet. 2001; 2:235-258; M. Kokoris, K. Dix, K. Moynihan, J. Mathis, B. Erwin, P. Grass, B. Hines and A. Duesterhoeft, “High-throughput SNP genotyping with the Masscode system,” Mol. Diagn. 2000 December; 5(4):329-340).

SNPs are identified by directly sequencing cDNA clones of the invention and by comparing the sequences with public and proprietary sequences. By comparing these cDNA clones with high quality proprietary or public sequences, seven SNPs were identified and two of them are linked (a deletion and a substitution). The transcripts or proteins with alternative alleles were designated as variants 158P3D2 v.3, v.4, v.5, v.6, v.7 and v.8. FIG. 10 shows the schematic alignment of the nucleotide variants. FIG. 11 shows the schematic alignment of protein variants, corresponding to nucleotide variants. Nucleotide variants that code for the same amino acid sequence as variant 1 are not shown in FIG. 11. These alleles of the SNPs, though shown separately here, can occur in different combinations (haplotypes) and in different transcript variants that contain the sequence context.

Example 7 Production of Recombinant 158P3D2 in Prokaryotic Systems

To express recombinant 158P3D2 and 158P3D2 variants in prokaryotic cells, the full or partial length 158P3D2 and 158P3D2 variant cDNA sequences can be cloned into any one of a variety of expression vectors known in the art. One or more of the following regions of 158P3D2 or 158P3D2 variants are expressed in these constructs, amino acids 1 to 328 of 158P3D2 (variant 1), amino acids 1-236 of variant 2a, amino acids 1-181 of variant 2b, amino acids 1-328 of variant 3, amino acids 1-328 of variant 4, amino acids 1-178 of variant 5a, amino acids 1-181 of variant 5b; or any 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30 or more contiguous amino acids from 158P3D2, variants, or analogs thereof.

A. In Vitro Transcription and Translation Constructs:

pCRII: To generate 158P3D2 sense and anti-sense RNA probes for RNA in situ investigations, pCRII constructs (Invitrogen, Carlsbad Calif.) are generated encoding either all or fragments of the 158P3D2 cDNA. The pCRII vector has Sp6 and T7 promoters flanking the insert to drive the transcription of 158P3D2 RNA for use as probes in RNA in situ hybridization experiments. These probes are used to analyze the cell and tissue expression of 158P3D2 at the RNA level. Transcribed 158P3D2 RNA representing the cDNA amino acid coding region of the 158P3D2 gene is used in in vitro translation systems such as the Tn™ Coupled Reticulolysate Sytem (Promega, Corp., Madison, Wis.) to synthesize 158P3D2 protein.

B. Bacterial Constructs:

pGEX Constructs: To generate recombinant 158P3D2 proteins in bacteria that are fused to the Glutathione S-transferase (GST) protein, all or parts of the T-fusion vector of the pGEX family (Amersham Pharmacia Biotech, Piscataway, N.J.). These constructs allow controlled expression of recombinant 158P3D2 protein sequences with GST fused at the amino-terminus and a six histidine epitope (6×His) at the carboxyl-terminus. The GST and 6×His tags permit purification of the recombinant fusion protein from induced bacteria with the appropriate affinity matrix and allow recognition of the fusion protein with anti-GST and anti-His antibodies. The 6×His tag is generated by adding 6 histidine codons to the cloning primer at the 3′ end, e.g., of the open reading frame (ORF). A proteolytic cleavage site, such as the PreScission™ recognition site in pGEX-6P-1, may be employed such that it permits cleavage of the GST tag from 158P3D2-related protein. The ampicillin resistance gene and pBR322 origin permits selection and maintenance of the pGEX plasmids in E. coli.

pMAL Constructs: To generate, in bacteria, recombinant 158P3D2 proteins that are fused to maltose-binding protein (MBP), all or parts of the 158P3D2 cDNA protein coding sequence are fused to the MBP gene by cloning into the pMAL-c2X and pMAL-p2X vectors (New England Biolabs, Beverly, Mass.). These constructs allow controlled expression of recombinant 158P3D2 protein sequences with MBP fused at the amino-terminus and a 6×His epitope tag at the carboxyl-terminus. The MBP and 6×His tags permit purification of the recombinant protein from induced bacteria with the appropriate affinity matrix and allow recognition of the fusion protein with anti-MBP and anti-His antibodies. The 6×His epitope tag is generated by adding 6 histidine codons to the 3′ cloning primer. A Factor Xa recognition site permits cleavage of the pMAL tag from 158P3D2. The pMAL-c2X and pMAL-p2X vectors are optimized to express the recombinant protein in the cytoplasm or periplasm respectively. Periplasm expression enhances folding of proteins with disulfide bonds.

pET Constructs: To express 158P3D2 in bacterial cells, all or parts of the 158P3D2 cDNA protein coding sequence are cloned into the pET family of vectors (Novagen, Madison, Wis.). These vectors allow tightly controlled expression of recombinant 158P3D2 protein in bacteria with and without fusion to proteins that enhance solubility, such as NusA and thioredoxin (Trx), and epitope tags, such as 6×His and S-Tag™ that aid purification and detection of the recombinant protein. For example, constructs are made utilizing pET NusA fusion system 43.1 such that regions of the 158P3D2 protein are expressed as amino-terminal fusions to NusA. In one embodiment, a NusA-fusion protein encompassing amino acids 412-328 of 158P3D2 with a C-terminal 6×His tag was expressed in E. Coli, purified by metal chelate affinity chromatography, and used as an immunogen for generation of antibodies.

C. Yeast Constructs:

pESC Constructs: To express 158P3D2 in the yeast species Saccharomyces cerevisiae for generation of recombinant protein and functional studies, all or parts of the 158P3D2 cDNA protein coding sequence are cloned into the pESC family of vectors each of which contain I of 4 selectable markers, HIS3, TRP1, LEU2, and URA3 (Stratagene, La Jolla, Calif.). These vectors allow controlled expression from the same plasmid of up to 2 different genes or cloned sequences containing either Flag™ or Myc epitope tags in the same yeast cell. This system is useful to confirm protein-protein interactions of 158P3D2. In addition, expression in yeast yields similar post-translational modifications, such as glycosylations and phosphorylations, that are found when expressed in eukaryotic cells.

pESP Constructs: To express 158P3D2 in the yeast species Saccharomyces pombe, all or parts of the 158P3D2 cDNA protein coding sequence are cloned into the pESP family of vectors. These vectors allow controlled high level of expression of a 158P3D2 protein sequence that is fused at either the amino terminus or at the carboxyl terminus to GST which aids purification of the recombinant protein. A Flag™ epitope tag allows defection of the recombinant protein with anti-Flag™ antibody.

Example 8 Production of Recombinant 158P3D2 in Eukaryotic Systems

A. Mammalian Constructs:

To express recombinant 158P3D2 in eukaryotic cells, the full or partial length 158P3D2 cDNA sequences were cloned into any one of a variety of expression vectors known in the art. One or more of the following regions of 158P3D2 were expressed in these constructs, amino acids 1 to 328, or any 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30 or more contiguous amino acids from 158P3D2, variants, or analogs thereof. In certain embodiments a region of 158P3D2 was expressed that encodes an amino acid not shared amongst at least variants.

The constructs were transfected into any one of a wide variety of mammalian cells such as 293T cells. Transfected 293T cell lysates can be probed with the anti-158P3D2 polyclonal serum, described herein.

pcDNA4/HisMax Constructs: To express 158P3D2 in mammalian cells, a 158P3D2 ORF, or portions thereof, of 158P3D2 are cloned into pcDNA4/HisMax Version A (Invitrogen, Carlsbad, Calif.). Protein expression is driven from the cytomegalovirus (CMV) promoter and the SP16 translational enhancer. The recombinant protein has Xpress™ and six histidine (6×His) epitopes fused to the amino-terminus. The pcDNA4/HisMax vector also contains the bovine growth hormone (BGH) polyadenylation signal and transcription termination sequence to enhance mRNA stability along with the SV40 origin for episomal replication and simple vector rescue in cell lines expressing the large T antigen. The Zeocin resistance gene allows for selection of mammalian cells expressing the protein and the ampicillin resistance gene and ColE1 origin permits selection and maintenance of the plasmid in E. coli.

pcDNA3.1/MycHis Constructs: To express 158P3D2 in mammalian cells, a 158P3D2 ORF, or portions thereof, of 158P3D2 with a consensus Kozak translation initiation site was cloned into pcDNA3.1/MycHis Version A (Invitrogen, Carlsbad, Calif.). Protein expression is driven from the cytomegalovirus (CMV) promoter. The recombinant proteins have the myc epitope and 6×His epitope fused to the carboxyl-terminus. The pcDNA3.1/MycHis vector also contains the bovine growth hormone (BGH) polyadenylation signal and transcription termination sequence to enhance mRNA stability, along with the SV40 origin for episomal replication and simple vector rescue in cell lines expressing the large T antigen. The Neomycin resistance gene can be used, as it allows for selection of mammalian cells expressing the protein and the ampicillin resistance gene and ColE1 origin permits selection and maintenance of the plasmid in E. coli. FIG. 19 shows expression of 158P3D2.pcDNA3.1/mychis in transiently transfected 293T cells.

pcDNA3.1/CT-GFP-TOPO Construct: To express 158P3D2 in mammalian cells and to allow detection of the recombinant proteins using fluorescence, a 158P3D2 ORF, or portions thereof, with a consensus Kozak translation initiation site are cloned into pcDNA3.1/CT-GFP-TOPO (Invitrogen, CA). Protein expression is driven from the cytomegalovirus (CMV) promoter. The recombinant proteins have the Green Fluorescent Protein (GFP) fused to the carboxyl-terminus facilitating non-invasive, in vivo detection and cell biology studies. The pcDNA3.1 CT-GFP-TOPO vector also contains the bovine growth hormone (BGH) polyadenylation signal and transcription termination sequence to enhance mRNA stability along with the SV40 origin for episomal replication and simple vector rescue in cell lines expressing the large T antigen. The Neomycin resistance gene allows for selection of mammalian cells that express the protein, and the ampicillin resistance gene and ColE1 origin permits selection and maintenance of the plasmid in E. coli. Additional constructs with an amino-terminal GFP fusion are made in pcDNA3.1/NT-GFP-TOPO spanning the entire length of a 158P3D2 protein.

PAPtag: A 158P3D2 ORF, or portions thereof, is cloned into pAPtag-5 (GenHunter Corp. Nashville, Tenn.). This construct generates an alkaline phosphatase fusion at the carboxyl-terminus of a 158P3D2 protein while fusing the IgGK signal sequence to the amino-terminus. Constructs are also generated in which alkaline phosphatase with an amino-terminal IgGK signal sequence is fused to the amino-terminus of a 158P3D2 protein. The resulting recombinant 158P3D2 proteins are optimized for secretion into the media of transfected mammalian cells and can be used to identify proteins such as ligands or receptors that interact with 158P3D2 proteins. Protein expression is driven from the CMV promoter and the recombinant proteins also contain myc and 6×His epitopes fused at the carboxyl-terminus that facilitates detection and purification. The Zeocin resistance gene present in the vector allows for selection of mammalian cells expressing the recombinant protein and the ampicillin resistance gene permits selection of the plasmid in E. coli.

ptag5: A 158P3D2 ORF, or portions thereof, is cloned into pTag-5. This vector is similar to pAPtag but without the alkaline phosphatase fusion. This construct generates 158P3D2 protein with an amino-terminal IgGK signal sequence and myc and 6×His epitope tags at the carboxyl-terminus that facilitate detection and affinity purification. The resulting recombinant 158P3D2 protein is optimized for secretion into the media of transfected mammalian cells, and is used as immunogen or ligand to identify proteins such as ligands or receptors that interact with the 158P3D2 proteins. Protein expression is driven from the CMV promoter. The Zeocin resistance gene present in the vector allows for selection of mammalian cells expressing the protein, and the ampicillin resistance gene permits selection of the plasmid in E. coli.

PsecFc: A 158P3D2 ORF, or portions thereof, is also cloned into psecFc. The psecFc vector was assembled by cloning the human immunoglobulin G1 (IgG) Fc (hinge, CH2, CH3 regions) into pSecTag2 (Invitrogen, California). This construct generates an IgG1 Fc fusion at the carboxyl-terminus of the 158P3D2 proteins, while fusing the IgGK signal sequence to N-terminus. 158P3D2 fusions utilizing the murine IgG1 Fc region are also used. The resulting recombinant 158P3D2 proteins are optimized for secretion into the media of transfected mammalian cells, and can be used as immunogens or to identify proteins such as ligands or receptors that interact with 158P3D2 protein. Protein expression is driven from the CMV promoter. The hygromycin resistance gene present in the vector allows for selection of mammalian cells that express the recombinant protein, and the ampicillin resistance gene permits selection of the plasmid in E. coli.

pSRα Constructs: To generate mammalian cell lines that express 158P3D2 constitutively, 158P3D2 ORF, or portions thereof, of 158P3D2 are cloned into pSRα constructs. Amphotropic and ecotropic retroviruses are generated by transfection of pSRα constructs into the 293T-10A1 packaging line or co-transfection of pSRα and a helper plasmid (containing deleted packaging sequences) into the 293 cells, respectively. The retrovirus is used to infect a variety of mammalian cell lines, resulting in the integration of the cloned gene, 158P3D2, into the host cell-lines. Protein expression is driven from a long terminal repeat (LTR). The Neomycin resistance gene present in the vector allows for selection of mammalian cells that express the protein, and the ampicillin resistance gene and ColE1 origin permit selection and maintenance of the plasmid in E. coli. The retroviral vectors can thereafter be used for infection and generation of various cell lines using, for example, PC3, NIH 3T3, TsuPr1, 293 or rat-1 cells.

Additional pSRα constructs are made that fuse an epitope tag such as the FLAG™ tag to the carboxyl-terminus of 158P3D2 sequences to allow detection using anti-Flag antibodies. For example, the FLAG™ sequence 5′ gat tac aag gat gac gac gat aag 3′ (SEQ. ID. No.: 6319) is added to cloning primer at the 3′ end of the ORF. Additional pSRα constructs are made to produce both amino-terminal and carboxyl-terminal GFP and myc/6×His fusion proteins of the full-length 158P3D2 proteins.

Additional Viral Vectors: Additional constructs are made for viral-mediated delivery and expression of 158P3D2. High virus titer leading to high level expression of 158P3D2 is achieved in viral delivery systems such as adenoviral vectors and herpes amplicon vectors. A 158P3D2 coding sequences or fragments thereof are amplified by PCR and subcloned into the AdEasy shuttle vector (Stratagene). Recombination and virus packaging are performed according to the manufacturer's instructions to generate adenoviral vectors. Alternatively, 158P3D2 coding sequences or fragments thereof are cloned into the HSV-1 vector (Imgenex) to generate herpes viral vectors. The viral vectors are thereafter used for infection of various cell lines such as PC3, NIH 3T3, 293 or rat-1 cells.

Regulated Expression Systems: To control expression of 158P3D2 in mammalian cells, coding sequences of 158P3D2, or portions thereof, are cloned into regulated mammalian expression systems such as the T-Rex System (Invitrogen), the GeneSwitch System (Invitrogen) and the tightly-regulated Ecdysone System (Sratagene). These systems allow the study of the temporal and concentration dependent effects of recombinant 158P3D2. These vectors are thereafter used to control expression of 158P3D2 in various cell lines such as PC3, NIH 3T3, 293 or rat-1 cells.

B. Baculovirus Expression Systems

To generate recombinant 158P3D2 proteins in a baculovirus expression system, 158P3D2 ORF, or portions thereof, are cloned into the baculovirus transfer vector pBlueBac 4.5 (Invitrogen), which provides a His-tag at the N-terminus. Specifically, pBlueBac-158P3D2 is co-transfected with helper plasmid pBac-N-Blue (Invitrogen) into SF9 (Spodoptera frugiperda) insect cells to generate recombinant baculovirus (see Invitrogen instruction manual for details). Baculovirus is then collected from cell supernatant and purified by plaque assay.

Recombinant 158P3D2 protein is then generated by infection of HighFive insect cells (Invitrogen) with purified baculovirus. Recombinant 158P3D2 protein can be detected using anti-158P3D2 or anti-His-tag antibody. 158P3D2 protein can be purified and used in various cell-based assays or as immunogen to generate polyclonal and monoclonal antibodies specific for 158P3D2.

Example 9 Antizenicity Profiles and Secondary Structure

FIG. 5A, FIG. 6A, FIG. 7A, FIG. 8A, and FIG. 9A depict graphically five amino acid profiles of the 158P3D2 variant 1 amino acid sequence; FIG. 5B, FIG. 6B, FIG. 7B, FIG. 8B, and FIG. 9B depict graphically five amino acid profiles of the 158P3D2 variant 2A amino acid sequence, and FIG. 5C, FIG. 6C, FIG. 7C, FIG. 8C, and FIG. 9C depict graphically five amino acid profiles of the 158P3D2 variant 5A amino acid sequence, each assessment available by accessing the ProtScale website on the ExPasy molecular biology server.

These profiles: FIG. 5, Hydrophilicity, (Hopp T. P., Woods K. R., 1981. Proc. Natl. Acad. Sci. U.S.A. 78:3824-3828); FIG. 6, Hydropathicity, (Kyte J., Doolittle R. F., 1982. J. Mol. Biol. 157:105-132); FIG. 7, Percentage Accessible Residues (Janin J., 1979 Nature 277:491-492); FIG. 8, Average Flexibility, (Bhaskaran R., and Ponnuswamy P. K., 1988. Int. J. Pept. Protein Res. 32:242-255); FIG. 9, Beta-turn (Deleage, G., Roux B. 1987 Protein Engineering 1:289-294); and optionally others available in the art, such as on the ProtScale website, were used to identify antigenic regions of the 158P3D2 protein. Each of the above amino acid profiles of 158P3D2 were generated using the following ProtScale parameters for analysis: 1) A window size of 9; 2) 100% weight of the window edges compared to the window center; and, 3) amino acid profile values normalized to lie between 0 and 1.

Hydrophilicity (FIG. 5), Hydropathicity (FIG. 6) and Percentage Accessible Residues (FIG. 7) profiles were used to determine stretches of hydrophilic amino acids (i.e., values greater than 0.5 on the Hydrophilicity and Percentage Accessible Residues profile, and values less than 0.5 on the Hydropathicity profile). Such regions are likely to be exposed to the aqueous environment, be present on the surface of the protein, and thus available for immune recognition, such as by antibodies.

Average Flexibility (FIG. 8) and Beta-turn (FIG. 9) profiles determine stretches of amino acids (i.e., values greater than 0.5 on the Beta-turn profile and the Average Flexibility profile) that are not constrained in secondary structures such as beta sheets and alpha helices. Such regions are also more likely to be exposed on the protein and thus accessible to immune recognition, such as by antibodies.

Antigenic sequences of the 158P3D2 protein and of the variant proteins indicated, e.g., by the profiles set forth in FIG. 5A-C, FIG. 6A-C, FIG. 7A-C, FIG. 8A-C, and/or FIG. 9A-C are used to prepare immunogens, either peptides or nucleic acids that encode them, to generate therapeutic and diagnostic anti-158P3D2 antibodies. The immunogen can be any 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 30, 35, 40, 45, 50 or more than 50 contiguous amino acids, or the corresponding nucleic acids that encode them, from the 158P3D2 protein or of 158P3D2 variants. In particular, peptide immunogens of the invention can comprise, a peptide region of at least 5 amino acids of FIG. 2 in any whole number increment up to 328 that includes an amino acid position having a value greater than 0.5 in the Hydrophilicity profile of FIG. 5; a peptide region of at least 5 amino acids of FIG. 2 in any whole number increment up to 328 that includes an amino acid position having a value less than 0.5 in the Hydropathicity profile of FIG. 6; a peptide region of at least 5 amino acids of FIG. 2 in any whole number increment up to 328 that includes an amino acid position having a value greater than 0.5 in the Percent Accessible Residues profile of FIG. 7; a peptide region of at least 5 amino acids of FIG. 2 in any whole number increment up to 328 that includes an amino acid position having a value greater than 0.5 in the Average Flexibility profile on FIG. 8; and, a peptide region of at least 5 amino acids of FIG. 2 in any whole number increment up to 328 that includes an amino acid position having a value greater than 0.5 in the Beta-turn profile of FIG. 9. Peptide immunogens of the invention can also comprise nucleic acids that encode any of the forgoing.

All immunogens of the invention, peptide or nucleic acid, can be embodied in human unit dose form, or comprised by a composition that includes a pharmaceutical excipient compatible with human physiology.

The secondary structure of 158P3D2 variant 1 and variants 2a and 5a, namely the predicted presence and location of alpha helices, extended strands, and random coils, is predicted from the primary amino acid sequence using the HNN—Hierarchical Neural Network method (Guermeur, 1997), accessed from the ExPasy molecular biology server. The analysis indicates that 158P3D2 variant 1 is composed 32.93% alpha helix, 18.29% extended strand, and 48.78% random coil (FIG. 12A), variant 2a is composed of 25.85% alpha helix, 18.22% extended strand, and 55.93% random coil (FIG. 12B), and variant 5a is composed of 9.55% alpha helix, 26.40% extended strand, and 64.04% random coil (FIG. 12C).

Analysis for the potential presence of transmembrane domains in 158P3D2 variant 1 was carried out using a variety of transmembrane prediction algorithms accessed from the ExPasy molecular biology server. The programs predict the presence of a single transmembrane domain in 158P3D2 variant 1. Shown graphically in FIGS. 12D and 12E are the results of analysis using the TMpred (FIG. 12D) and TMHMM (FIG. 12E) prediction programs depicting the location of the transmembrane domain. The results of each program, namely the amino acids encoding the transmembrane domain are summarized in Table XXII. Variants 2b, 3, 4, and 5b, also contain the amino acids predicted to encode the transmembrane domain. No transmembrane domains are predicted in variants 2a and 5a.

Example 10 Generation of 158P3D2 Polyclonal Antibodies

Polyclonal antibodies can be raised in a mammal, for example, by one or more injections of an immunizing agent and, if desired, an adjuvant. Typically, the immunizing agent and/or adjuvant will be injected in the mammal by multiple subcutaneous or intraperitoneal injections. In addition to immunizing with the full length 158P3D2 protein, computer algorithms are employed in design of immunogens that, based on amino acid sequence analysis contain characteristics of being antigenic and available for recognition by the immune system of the immunized host (see Example 9). Such regions would be predicted to be hydrophilic, flexible, in beta-turn conformations, and be exposed on the surface of the protein (see, e.g., FIG. 5A-C, FIG. 6 A-C, FIG. 7 A-C, FIG. 8 A-C, or FIG. 9 A-C for amino acid profiles that indicate such regions of 158P3D2 and variants).

For example, 158P3D2 recombinant bacterial fusion proteins or peptides containing hydrophilic, flexible, beta-turn regions of the 158P3D2, such as regions amino terminal to the predicted transmembrane domain of variant 1 (predicted to be extracellular), are used as antigens to generate polyclonal antibodies in New Zealand White rabbits. For example, such regions include, but are not limited to, amino acids 1-25, amino acids 37-54, amino acids 60-73, and amino acids 187-225. It is useful to conjugate the immunizing agent to a protein known to be immunogenic in the mammal being immunized. Examples of such immunogenic proteins include, but are not limited to, keyhole limpet hemocyanin (KLH), serum albumin, bovine thyroglobulin, and soybean trypsin inhibitor. In one embodiment, a peptide encoding amino acids 200-225 of 158P3D2 is conjugated to KLH and used to immunize the rabbit. Alternatively the immunizing agent may include all or portions of the 158P3D2 protein, analogs or fusion proteins thereof. For example, the 158P3D2 amino acid sequence can be fused using recombinant DNA techniques to any one of a variety of fusion protein partners that are well known in the art, such as glutathione-S-transferase (GST) and HIS tagged fusion proteins. Such fusion proteins are purified from induced bacteria using the appropriate affinity matrix.

In one embodiment, a GST-fusion protein encoding the predicted extracellular domain, amino acids 1-291, is produced and purified and used as immunogen. Other recombinant bacterial fusion proteins that may be employed include maltose binding protein, LacZ, thioredoxin, NusA, or an immunoglobulin constant region (see Example 7 and Current Protocols In Molecular Biology, Volume 2, Unit 16, Frederick M. Ausubul et al. eds., 1995; Linsley, P. S., Brady, W., Urnes, M., Grosmaire, L., Damle, N., and Ledbetter, L. (1991) J. Exp. Med. 174, 561-566).

In addition to bacterial derived fusion proteins, mammalian expressed protein antigens are also used. These antigens are expressed from mammalian expression vectors such as the Tag5 and Fc-fusion vectors (see Example 8), and retain post-translational modifications such as glycosylations found in native protein.

In one embodiment, amino acids 185-225 is cloned into the Tag5 mammalian secretion vector. In another embodiment, the predicted extracellular domain, amino acids 1-291 is cloned into the Tag5 expression vector. The recombinant proteins are purified by metal chelate chromatography from tissue culture supernatants of 293T cells stably expressing the recombinant vector. The purified Tag5 158P3D2 proteins are then individually used as immunogens.

During the immunization protocol, it is useful to mix or emulsify the antigen in adjuvants that enhance the immune response of the host animal. Examples of adjuvants include, but are not limited to, complete Freund's adjuvant (CFA) and MPL-TDM adjuvant (monophosphoryl Lipid A, synthetic trehalose dicorynomycolate).

In a typical protocol, rabbits are initially immunized subcutaneously with up to 200 μg, typically 100-200 μg, of fusion protein or peptide conjugated to KLH mixed in complete Freund's adjuvant (CFA). Rabbits are then injected subcutaneously every two weeks with up to 200 μg, typically 100-200 μg, of the immunogen in incomplete Freund's adjuvant (IFA). Test bleeds are taken approximately 7-10 days following each immunization and used to monitor the titer of the antiserum by ELISA.

To test reactivity and specificity of immune serum, such as the rabbit serum derived from immunization with Tag5 158P3D2 encoding amino acids 1-291, the full-length 158P3D2 cDNA is cloned into pcDNA 3.1 myc-his expression vector (Invitrogen, see Example 7). After transfection of the constructs into 293T cells, cell lysates are probed with the anti-158P3D2 serum and with anti-His antibody (Santa Cruz Biotechnologies, Santa Cruz, Calif.) to determine specific reactivity to denatured 158P3D2 protein using the Western blot technique. Shown in FIG. 19 is expression of Myc His tagged 158P3D2 protein in 293T cells as detected by Western blot with anti-His antibody. The immune serum is then tested by the Western blot technique against 293T-158P3D2 cells. In addition, the immune serum is tested by fluorescence microscopy, flow cytometry and immunoprecipitation against 293T and other recombinant 158P3D2-expressing cells to determine specific recognition of native protein. Western blot, immunoprecipitation, fluorescent microscopy, and flow cytometric techniques using cells that endogenously express 158P3D2 are also carried out to test reactivity and specificity.

Anti-serum from rabbits immunized with 158P3D2 fusion proteins, such as GST and MBP fusion proteins, are purified by depletion of antibodies reactive to the fusion partner sequence by passage over an affinity column containing the fusion partner either alone or in the context of an irrelevant fusion protein. For example, antiserum derived from a GST-158P3D2 fusion protein encoding amino acids 1-291 is first purified by passage over a column of GST protein covalently coupled to AffiGel matrix (BioRad, Hercules, Calif.). The antiserum is then affinity purified by passage over a column composed of a MBP-fusion protein also encoding amino acids 1-291 covalently coupled to Affigel matrix. The serum is then further purified by protein G affinity chromatography to isolate the IgG fraction. Sera from other His-tagged antigens and peptide immunized rabbits as well as fusion partner depleted sera are affinity purified by passage over a column matrix composed of the original protein immunogen or free peptide.

Example 11 Generation of 158P3D2 Monoclonal Antibodies (mAbs)

In one embodiment, therapeutic mAbs to 158P3D2 comprise those that react with epitopes of the protein that would disrupt or modulate the biological function of 158P3D2, for example those that would disrupt its interaction with ligands and binding partners. Therapeutic mAbs also comprise those that specifically bind epitopes of 158P3D2 exposed on the cell surface and thus are useful in targeting mAb-toxin conjugates. Immunogens for generation of such mAbs include those designed to encode or contain the entire 158P3D2 protein, regions of the 158P3D2 protein predicted to be antigenic from computer analysis of the amino acid sequence (see, e.g., FIG. 5A-C, FIG. 6 A-C, FIG. 7 A-C, FIG. 8 A-C, or FIG. 9 A-C, and Example 9) such as regions in the extracellular domain of variant 1. Immunogens include peptides, recombinant bacterial proteins, and mammalian expressed Tag 5 proteins and human and murine IgG FC fusion proteins. In addition, cells expressing high levels of 158P3D2, such as 293T-158P3D2 or 300.19-158P3D2 murine Pre-B cells, are used to immunize mice.

To generate mAbs to 158P3D2, mice are first immunized intraperitoneally (IP) with, typically, 10-50 μg of protein immunogen or 10⁷ 158P3D2-expressing cells mixed in complete Freund's adjuvant. Mice are then subsequently immunized IP every 2-4 weeks with, typically, 10-50 μg of protein immunogen or 10⁷ cells mixed in incomplete Freund's adjuvant. Alternatively, MPL-TDM adjuvant is used in immunizations. In addition to the above protein and cell-based immunization strategies, a DNA-based immunization protocol is employed in which a mammalian expression vector encoding 158P3D2 sequence is used to immunize mice by direct injection of the plasmid DNA. For example, amino acids 1-291 is cloned into the Tag5 mammalian secretion vector and the recombinant vector is used as immunogen. In another example the same amino acids are cloned into an Fc-fusion secretion vector in which the 158P3D2 sequence is fused at the amino-terminus to an IgK leader sequence and at the carboxyl-terminus to the coding sequence of the human or murine IgG Fc region. This recombinant vector is then used as immunogen. The plasmid immunization protocols are used in combination with purified proteins expressed from the same vector and with cells expressing 158P3D2.

During the immunization protocol, test bleeds are taken 7-10 days following an injection to monitor titer and specificity of the immune response. Once appropriate reactivity and specificity is obtained as determined by ELISA, Western blotting, immunoprecipitation, fluorescence microscopy, and flow cytometric analyses, fusion and hybridoma generation is then carried out with established procedures well known in the art (see, e.g., Harlow and Lane, 1988).

In one embodiment for generating 158P3D2 monoclonal antibodies, a Tag5-158P3D2 antigen encoding amino acids 1-291, the predicted extracellular domain, is expressed and purified from stably transfected 293T cells. Balb C mice are initially immunized intraperitoneally with 25 μg of the Tag5-158P3D2 protein mixed in complete Freund's adjuvant. Mice are subsequently immunized every two weeks with 25 μg of the antigen mixed in incomplete Freund's adjuvant for a total of three immunizations. ELISA using the Tag5 antigen determines the titer of serum from immunized mice. Reactivity and specificity of serum to full length 158P3D2 protein is monitored by Western blotting, immunoprecipitation and flow cytometry using 293T cells transfected with an expression vector encoding the 158P3D2 cDNA (see e.g., Example 8). Other recombinant 158P3D2-expressing cells or cells endogenously expressing 158P3D2 are also used. Mice showing the strongest reactivity are rested and given a final injection of Tag5 antigen in PBS and then sacrificed four days later. The spleens of the sacrificed mice are harvested and fused to SPO/2 myeloma cells using standard procedures (Harlow and Lane, 1988). Supernatants from HAT selected growth wells are screened by ELISA, Western blot, immunoprecipitation, fluorescent microscopy, and flow cytometry to identify 158P3D2 specific antibody-producing clones.

Monoclonal antibodies are also derived that react only with specific 158P3D2 variants, such as variants 2a and 5a. To this end, immunogens are designed to encode amino acid regions specific to the respective variant. For example, a Tag5 immunogen is encoding amino acids 1-236 of variant 2a is produced, purified, and used to immunize mice to generate hybridomas. In another example, a Tag5 immunogen encoding amino acids 130-178 of variant 5a is produced, purified, and used as immunogen. Monoclonal antibodies raised to these immunogens are then screened for reactivity to cells expressing the respective variants but not to other 158P3D2 variants. These strategies for raising 158P3D2 variant specific monoclonal antibodies are also applied to polyclonal reagents described in Example 10.

The binding affinity of a 158P3D2 monoclonal antibody is determined using standard technologies. Affinity measurements quantify the strength of antibody to epitope binding and are used to help define which 158P3D2 monoclonal antibodies preferred for diagnostic or therapeutic use, as appreciated by one of skill in the art. The BIAcore system (Uppsala, Sweden) is a preferred method for determining binding affinity. The BIAcore system uses surface plasmon resonance (SPR, Welford K. 1991, Opt. Quant. Elect. 23:1; Morton and Myszka, 1998, Methods in Enzymology 295: 268) to monitor biomolecular interactions in real time. BIAcore analysis conveniently generates association rate constants, dissociation rate constants, equilibrium dissociation constants, and affinity constants.

Example 12 HLA Class I and Class II Binding Assays

HLA class I and class II binding assays using purified HLA molecules are performed in accordance with disclosed protocols (e.g., PCT publications WO 94/20127 and WO 94/03205; Sidney et al., Current Protocols in Immunology 18.3.1 (1998); Sidney, et al., J. Immunol. 154:247 (1995); Sette, et al., Mol. Immunol. 31:813 (1994)). Briefly, purified MHC molecules (5 to 500 nM) are incubated with various unlabeled peptide inhibitors and 1-10 nM ¹²⁵I-radiolabeled probe peptides as described. Following incubation, MHC-peptide complexes are separated from free peptide by gel filtration and the fraction of peptide bound is determined. Typically, in preliminary experiments, each MHC preparation is titered in the presence of fixed amounts of radiolabeled peptides to determine the concentration of HLA molecules necessary to bind 10-20% of the total radioactivity. All subsequent inhibition and direct binding assays are performed using these HLA concentrations.

Since under these conditions [label]<[HLA] and IC₅₀>[HLA], the measured IC₅₀ values are reasonable approximations of the true K_(D) values. Peptide inhibitors are typically tested at concentrations ranging from 120 μg/ml to 1.2 ng/ml, and are tested in two to four completely independent experiments. To allow comparison of the data obtained in different experiments, a relative binding figure is calculated for each peptide by dividing the IC₅₀ of a positive control for inhibition by the IC₅₀ for each tested peptide (typically unlabeled versions of the radiolabeled probe peptide). For database purposes, and inter-experiment comparisons, relative binding values are compiled. These values can subsequently be converted back into IC₅₀ nM values by dividing the IC₅₀ nM of the positive controls for inhibition by the relative binding of the peptide of interest. This method of data compilation is accurate and consistent for comparing peptides that have been tested on different days, or with different lots of purified MHC.

Binding assays as outlined above may be used to analyze HLA supermotif and/or HLA motif-bearing peptides.

Example 13 Identification of HLA Supermotif- and Motif-Bearing CTL Candidate Epitopes

HLA vaccine compositions of the invention can include multiple epitopes. The multiple epitopes can comprise multiple HLA supermotifs or motifs to achieve broad population coverage. This example illustrates the identification and confirmation of supermotif- and motif-bearing epitopes for the inclusion in such a vaccine composition. Calculation of population coverage is performed using the strategy described below.

Computer Searches and Algorithms for Identification of Supermotif and/or Motif-Bearing Epitopes

The searches performed to identify the motif-bearing peptide sequences in Example 9 and Tables V-XIX employ the protein sequence data from the gene product of 158P3D2 set forth in FIGS. 2 and 3.

Computer searches for epitopes bearing HLA Class I or Class II supermotifs or motifs are performed as follows. All translated 158P3D2 protein sequences are analyzed using a text string search software program to identify potential peptide sequences containing appropriate HLA binding motifs; such programs are readily produced in accordance with information in the art in view of known motif/supermotif disclosures. Furthermore, such calculations can be made mentally.

Identified A2-, A3-, and DR-supermotif sequences are scored using polynomial algorithms to predict their capacity to bind to specific HLA-Class I or Class II molecules. These polynomial algorithms account for the impact of different amino acids at different positions, and are essentially based on the premise that the overall affinity (or AG) of peptide-HLA molecule interactions can be approximated as a linear polynomial function of the type:

“ΔG”=a ₁ ×a ₂ ×a _(3i) . . . ×a _(ni)

where a_(ji) is a coefficient which represents the effect of the presence of a given amino acid (j) at a given position (i) along the sequence of a peptide of n amino acids. The crucial assumption of this method is that the effects at each position are essentially independent of each other (i.e., independent binding of individual side-chains). When residuej occurs at position i in the peptide, it is assumed to contribute a constant amount j_(i) to the free energy of binding of the peptide irrespective of the sequence of the rest of the peptide.

The method of derivation of specific algorithm coefficients has been described in Gulukota et al., J. Mol. Biol. 267:1258-126, 1997; (see also Sidney et al., Human Immunol. 45:79-93, 1996; and Southwood et al., J. Immunol. 160:3363-3373, 1998). Briefly, for all i positions, anchor and non-anchor alike, the geometric mean of the average relative binding (ARB) of all peptides carrying j is calculated relative to the remainder of the group, and used as the estimate of j_(i). For Class II peptides, if multiple alignments are possible, only the highest scoring alignment is utilized, following an iterative procedure. To calculate an algorithm score of a given peptide in a test set, the ARB values corresponding to the sequence of the peptide are multiplied. If this product exceeds a chosen threshold, the peptide is predicted to bind. Appropriate thresholds are chosen as a function of the degree of stringency of prediction desired.

Selection of HLA-A2 Supertype Cross-Reactive Peptides

Protein sequences from 158P3D2 are scanned utilizing motif identification software, to identify 8-, 9-10- and 1-mer sequences containing the HLA-A2-supermotif main anchor specificity. Typically, these sequences are then scored using the protocol described above and the peptides corresponding to the positive-scoring sequences are synthesized and tested for their capacity to bind purified HLA-A*0201 molecules in vitro (HLA-A*0201 is considered a prototype A2 supertype molecule).

These peptides are then tested for the capacity to bind to additional A2-supertype molecules (A*0202, A*0203, A*0206, and A*6802). Peptides that bind to at least three of the five A2-supertype alleles tested are typically deemed A2-supertype cross-reactive binders. Preferred peptides bind at an affinity equal to or less than 500 nM to three or more HLA-A2 supertype molecules.

Selection of HLA-A3 Supermotif-Bearing Epitopes

The 158P3D2 protein sequence(s) scanned above is also examined for the presence of peptides with the HLA-A3-supermotif primary anchors. Peptides corresponding to the HLA A3 supermotif-bearing sequences are then synthesized and tested for binding to HLA-A*0301 and HLA-A* 1101 molecules, the molecules encoded by the two most prevalent A3-supertype alleles. The peptides that bind at least one of the two alleles with binding affinities of <500 nM, often ≦200 nM, are then tested for binding cross-reactivity to the other common A3-supertype alleles (e.g., A*3101, A*3301, and A*6801) to identify those that can bind at least three of the five HLA-A3-supertype molecules tested.

Selection of HLA-B7 Supermotif Bearing Epitopes

The 158P3D2 protein(s) scanned above is also analyzed for the presence of 8-, 9-10-, or 11-mer peptides with the HLA-B7-supermotif. Corresponding peptides are synthesized and tested for binding to HLA-B*0702, the molecule encoded by the most common B7-supertype allele (i.e., the prototype B7 supertype allele). Peptides binding B*0702 with IC₅₀ of <500 nM are identified using standard methods. These peptides are then tested for binding to other common B7-supertype molecules (e.g., B*3501, B*5101, B*5301, and B*5401). Peptides capable of binding to three or more of the five B7-supertype alleles tested are thereby identified.

Selection of A1 and A24 Motif-Bearing Epitopes

To further increase population coverage, HLA-A1 and -A24 epitopes can also be incorporated into vaccine compositions. An analysis of the 158P3D2 protein can also be performed to identify HLA-A1- and A24-motif-containing sequences.

High affinity and/or cross-reactive binding epitopes that bear other motif and/or supermotifs are identified using analogous methodology.

Example 14 Confirmation of Immunogenicity

Cross-reactive candidate CTL A2-supermotif-bearing peptides that are identified as described herein are selected to confirm in vitro immunogenicity. Confirmation is performed using the following methodology:

Target Cell Lines for Cellular Screening:

The 0.221A2.1 cell line, produced by transferring the HLA-A2.1 gene into the HLA-A, -B, -C null mutant human B-lymphoblastoid cell line 721.221, is used as the peptide-loaded target to measure activity of HLA-A2.1-restricted CTL. This cell line is grown in RPMI-1640 medium supplemented with antibiotics, sodium pyruvate, nonessential amino acids and 10% (v/v) heat inactivated FCS. Cells that express an antigen of interest, or transfectants comprising the gene encoding the antigen of interest, can be used as target cells to confirm the ability of peptide-specific CTLs to recognize endogenous antigen.

Primary CTL Induction Cultures:

Generation of Dendritic Cells (DC): PBMCs are thawed in RPMI with 30 μg/ml DNAse, washed twice and resuspended in complete medium (RPMI-1640 plus 5% AB human serum, non-essential amino acids, sodium pyruvate, L-glutamine and penicillin/streptomycin). The monocytes are purified by plating 10×10⁶ PBMC/well in a 6-well plate. After 2 hours at 37° C., the non-adherent cells are removed by gently shaking the plates and aspirating the supernatants. The wells are washed a total of three times with 3 ml RPMI to remove most of the non-adherent and loosely adherent cells. Three ml of complete medium containing 50 ng/ml of GM-CSF and 1,000 U/ml of IL-4 are then added to each well. TNFα is added to the DCs on day 6 at 75 ng/ml and the cells are used for CTL induction cultures on day 7.

Induction of CTL with DC and Peptide: CD8+ T-cells are isolated by positive selection with Dynal immunomagnetic beads (Dynabeads® M-450) and the Detacha-Bead® reagent. Typically about 200-250×10⁶ PBMC are processed to obtain 24×10⁶ CD8⁺ T-cells (enough for a 48-well plate culture). Briefly, the PBMCs are thawed in RPMI with 30 μg/ml DNAse, washed once with PBS containing 1% human AB serum and resuspended in PBS/1% AB serum at a concentration of 20×10⁶ cells/ml. The magnetic beads are washed 3 times with PBS/AB serum, added to the cells (14011 beads/20×10⁶ cells) and incubated for 1 hour at 4° C. with continuous mixing. The beads and cells are washed 4× with PBS/AB serum to remove the nonadherent cells and resuspended at 100×10⁶ cells/ml (based on the original cell number) in PBS/AB serum containing 100 μl/ml Detacha-Bead® reagent and 30 μg/ml DNAse. The mixture is incubated for 1 hour at room temperature with continuous mixing. The beads are washed again with PBS/AB/DNAse to collect the CD8+ T-cells. The DC are collected and centrifuged at 1300 rpm for 5-7 minutes, washed once with PBS with 1% BSA, counted and pulsed with 40 μg/ml of peptide at a cell concentration of 1-2×10⁶/ml in the presence of 3 μg/ml β₂-microglobulin for 4 hours at 20° C. The DC are then irradiated (4,200 rads), washed 1 time with medium and counted again.

Setting up induction cultures: 0.25 ml cytokine-generated DC (at 1×10⁵ cells/ml) are co-cultured with 0.25 ml of CD8+ T-cells (at 2×10⁶ cell/ml) in each well of a 48-well plate in the presence of 10 ng/ml of IL-7. Recombinant human IL-10 is added the next day at a final concentration of 10 ng/ml and rhuman IL-2 is added 48 hours later at 10 IU/ml.

Restimulation of the induction cultures with peptide-pulsed adherent cells: Seven and fourteen days after the primary induction, the cells are restimulated with peptide-pulsed adherent cells. The PBMCs are thawed and washed twice with RPMI and DNAse. The cells are resuspended at 5×10⁶ cells/ml and irradiated at ˜4200 rads. The PBMCs are plated at 2×10⁶ in 0.5 ml complete medium per well and incubated for 2 hours at 37° C. The plates are washed twice with RPMI by tapping the plate gently to remove the nonadherent cells and the adherent cells pulsed with 10 μg/ml of peptide in the presence of 3 μg/ml β₂ microglobulin in 0.25 ml RPMI/5% AB per well for 2 hours at 37° C. Peptide solution from each well is aspirated and the wells are washed once with RPMI. Most of the media is aspirated from the induction cultures (CD8+ cells) and brought to 0.5 ml with fresh media. The cells are then transferred to the wells containing the peptide-pulsed adherent cells. Twenty four hours later recombinant human IL-10 is added at a final concentration of 10 ng/ml and recombinant human IL2 is added the next day and again 2-3 days later at 50 IU/ml (Tsai et al., Critical Reviews in Immunology 18(1-2):65-75, 1998). Seven days later, the cultures are assayed for CTL activity in a ⁵¹Cr release assay. In some experiments the cultures are assayed for peptide-specific recognition in the in situ IFNγ ELISA at the time of the second restimulation followed by assay of endogenous recognition 7 days later. After expansion, activity is measured in both assays for a side-by-side comparison.

Measurement of CTL Lytic Activity by ⁵¹Cr Release.

Seven days after the second restimulation, cytotoxicity is determined in a standard (5 hr) ⁵¹Cr release assay by assaying individual wells at a single E:T. Peptide-pulsed targets are prepared by incubating the cells with 10 μg/ml peptide overnight at 37° C.

Adherent target cells are removed from culture flasks with trypsin-EDTA. Target cells are labeled with 200 μCi of ⁵¹Cr sodium chromate (Dupont, Wilmington, Del.) for 1 hour at 37° C. Labeled target cells are resuspended at 10⁶ per ml and diluted 1:10 with K562 cells at a concentration of 3.3×10⁶/ml (an NK-sensitive erythroblastoma cell line used to reduce non-specific lysis). Target cells (100 μl) and effectors (100 μl) are plated in 96 well round-bottom plates and incubated for 5 hours at 37° C. At that time, 100 μl of supernatant are collected from each well and percent lysis is determined according to the formula:

[(cpm of the test sample−cpm of the spontaneous ⁵¹Cr release sample)/(cpm of the maximal ⁵¹Cr release sample−cpm of the spontaneous ⁵¹Cr release sample)]×100.

Maximum and spontaneous release are determined by incubating the labeled targets with 1% Triton X-100 and media alone, respectively. A positive culture is defined as one in which the specific lysis (sample-background) is 10% or higher in the case of individual wells and is 15% or more at the two highest E:T ratios when expanded cultures are assayed.

In Situ Measurement of Human IFNγ Production as an Indicator of Peptide-Specific and Endogenous Recognition

Immulon 2 plates are coated with mouse anti-human IFNγ monoclonal antibody (4 μg/ml 0.1M NaHCO₃, pH 8.2) overnight at 4° C. The plates are washed with Ca²⁺, Mg²⁺-free PBS/0.05% Tween 20 and blocked with PBS/10% FCS for two hours, after which the CTLs (100 μl/well) and targets (100 μl/well) are added to each well, leaving empty wells for the standards and blanks (which received media only). The target cells, either peptide-pulsed or endogenous targets, are used at a concentration of 1×10⁶ cells/ml. The plates are incubated for 48 hours at 37° C. with 5% CO₂.

Recombinant human IFN-gamma is added to the standard wells starting at 400 pg or 1200 pg/100 microliter/well and the plate incubated for two hours at 37° C. The plates are washed and 100 μl of biotinylated mouse anti-human IFN-gamma monoclonal antibody (2 microgram/ml in PBS/3% FCS/0.05% Tween 20) are added and incubated for 2 hours at room temperature. After washing again, 100 microliter HRP-streptavidin (1:4000) are added and the plates incubated for one hour at room temperature. The plates are then washed 6× with wash buffer, 100 microliter/well developing solution (TMB 1:1) are added, and the plates allowed to develop for 5-15 minutes. The reaction is stopped with 50 microliter/well 1M H₃PO₄ and read at OD450. A culture is considered positive if it measured at least 50 pg of IFN-gamma/well above background and is twice the background level of expression.

CTL Expansion.

Those cultures that demonstrate specific lytic activity against peptide-pulsed targets and/or tumor targets are expanded over a two week period with anti-CD3. Briefly, 5×10⁴ CD8+ cells are added to a T25 flask containing the following: 1×10⁶ irradiated (4,200 rad) PBMC (autologous or allogeneic) per ml, 2×10⁵ irradiated (8,000 rad) EBV-transformed cells per ml, and OKT3 (anti-CD3) at 30 ng per ml in RPMI-1640 containing 10% (v/v) human AB serum, non-essential amino acids, sodium pyruvate, 25 μM 2-mercaptoethanol, L-glutamine and penicillin/streptomycin. Recombinant human IL2 is added 24 hours later at a final concentration of 200 IU/ml and every three days thereafter with fresh media at 50 IU/ml. The cells are split if the cell concentration exceeds 1×10⁶/ml and the cultures are assayed between days 13 and 15 at E:T ratios of 30, 10, 3 and 1:1 in the ⁵¹Cr release assay or at 1×10⁶/ml in the in situ IFNγ assay using the same targets as before the expansion.

Cultures are expanded in the absence of anti-CD3⁺ as follows. Those cultures that demonstrate specific lytic activity against peptide and endogenous targets are selected and 5×10⁴ CD8⁺ cells are added to a T25 flask containing the following: 1×10⁶ autologous PBMC per ml which have been peptide-pulsed with 10 μg/ml peptide for two hours at 37° C. and irradiated (4,200 rad); 2×10⁵ irradiated (8,000 rad) EBV-transformed cells per ml RPMI-1640 containing 10% (v/v) human AB serum, non-essential AA, sodium pyruvate, 25 mM 2-ME, L-glutamine and gentamicin.

Immunogenicity of A2 Supermotif-Bearing Peptides

A2-supermotif cross-reactive binding peptides are tested in the cellular assay for the ability to induce peptide-specific CTL in normal individuals. In this analysis, a peptide is typically considered to be an epitope if it induces peptide-specific CTLs in at least individuals, and preferably, also recognizes the endogenously expressed peptide.

Immunogenicity can also be confirmed using PBMCs isolated from patients bearing a tumor that expresses 158P3D2. Briefly, PBMCs are isolated from patients, re-stimulated with peptide-pulsed monocytes and assayed for the ability to recognize peptide-pulsed target cells as well as transfected cells endogenously expressing the antigen.

Evaluation of A*03/A11 Immunogenicity

HLA-A3 supermotif-bearing cross-reactive binding peptides are also evaluated for immunogenicity using methodology analogous for that used to evaluate the immunogenicity of the HLA-A2 supermotif peptides.

Evaluation of B7 Immunogenicity

Immunogenicity screening of the B7-supertype cross-reactive binding peptides identified as set forth herein are confirmed in a manner analogous to the confirmation of A2- and A3-supermotif-bearing peptides.

Peptides bearing other supermotifs/motifs, e.g., HLA-A 1, HLA-A24 etc. are also confirmed using similar methodology

Example 15 Implementation of the Extended Supermotif to Improve the Binding Capacity of Native Epitopes by Creating Analogs

HLA motifs and supermotifs (comprising primary and/or secondary residues) are useful in the identification and preparation of highly cross-reactive native peptides, as demonstrated herein. Moreover, the definition of HLA motifs and supermotifs also allows one to engineer highly cross-reactive epitopes by identifying residues within a native peptide sequence which can be analoged to confer upon the peptide certain characteristics, e.g. greater cross-reactivity within the group of HLA molecules that comprise a supertype, and/or greater binding affinity for some or all of those HLA molecules. Examples of analoging peptides to exhibit modulated binding affinity are set forth in this example.

Analoging at Primary Anchor Residues

Peptide engineering strategies are implemented to further increase the cross-reactivity of the epitopes. For example, the main anchors of A2-supermotif-bearing peptides are altered, for example, to introduce a preferred L, I, V, or M at position 2, and I or V at the C-terminus.

To analyze the cross-reactivity of the analog peptides, each engineered analog is initially tested for binding to the prototype A2 supertype allele A*0201, then, if A*0201 binding capacity is maintained, for A2-supertype cross-reactivity.

Alternatively, a peptide is confirmed as binding one or all supertype members and then analoged to modulate binding affinity to any one (or more) of the supertype members to add population coverage.

The selection of analogs for immunogenicity in a cellular screening analysis is typically further restricted by the capacity of the parent wild type (WT) peptide to bind at least weakly, i.e., bind at an IC₅₀ of 5000 nM or less, to three of more A2 supertype alleles. The rationale for this requirement is that the WT peptides must be present endogenously in sufficient quantity to be biologically relevant. Analoged peptides have been shown to have increased immunogenicity and cross-reactivity by T cells specific for the parent epitope (see, e.g., Parkhurst et al., J. Immunol. 157:2539, 1996; and Pogue et al., Proc. Natl. Acad. Sci. USA 92:8166, 1995).

In the cellular screening of these peptide analogs, it is important to confirm that analog-specific CTLs are also able to recognize the wild-type peptide and, when possible, target cells that endogenously express the epitope.

Analoging of HLA-A3 and B7-Supermotif-Bearing Peptides

Analogs of HLA-A3 supermotif-bearing epitopes are generated using strategies similar to those employed in analoging HLA-A2 supermotif-bearing peptides. For example, peptides binding to ⅗ of the A3-supertype molecules are engineered at primary anchor residues to possess a preferred residue (V, S, M, or A) at position 2.

The analog peptides are then tested for the ability to bind A*03 and A*11 (prototype A3 supertype alleles). Those peptides that demonstrate ≦500 nM binding capacity are then confirmed as having A3-supertype cross-reactivity.

Similarly to the A2- and A3-motif bearing peptides, peptides binding 3 or more B7-supertype alleles can be improved, where possible, to achieve increased cross-reactive binding or greater binding affinity or binding half life. B7 supermotif-bearing peptides are, for example, engineered to possess a preferred residue (V, I, L, or F) at the C-terminal primary anchor position, as demonstrated by Sidney et al. (J. Immunol. 157:3480-3490, 1996).

Analoging at primary anchor residues of other motif and/or supermotif-bearing epitopes is performed in a like manner.

The analog peptides are then be confirmed for immunogenicity, typically in a cellular screening assay. Again, it is generally important to demonstrate that analog-specific CTLs are also able to recognize the wild-type peptide and, when possible, targets that endogenously express the epitope.

Analoging at Secondary Anchor Residues

Moreover, HLA supermotifs are of value in engineering highly cross-reactive peptides and/or peptides that bind HLA molecules with increased affinity by identifying particular residues at secondary anchor positions that are associated with such properties. For example, the binding capacity of a B7 supermotif-bearing peptide with an F residue at position 1 is analyzed. The peptide is then analoged to, for example, substitute L for F at position 1. The analoged peptide is evaluated for increased binding affinity, binding half life and/or increased cross-reactivity. Such a procedure identifies analoged peptides with enhanced properties.

Engineered analogs with sufficiently improved binding capacity or cross-reactivity can also be tested for immunogenicity in HLA-B7-transgenic mice, following for example, IFA immunization or lipopeptide immunization. Analoged peptides are additionally tested for the ability to stimulate a recall response using PBMC from patients with 158P3D2-expressing tumors.

Other Analoging Strategies

Another form of peptide analoging, unrelated to anchor positions, involves the substitution of a cysteine with α-amino butyric acid. Due to its chemical nature, cysteine has the propensity to form disulfide bridges and sufficiently alter the peptide structurally so as to reduce binding capacity. Substitution of α-amino butyric acid for cysteine not only alleviates this problem, but has been shown to improve binding and crossbinding capabilities in some instances (see, e.g., the review by Sette et al., In: Persistent Viral Infections, Eds. R. Ahmed and 1. Chen, John Wiley & Sons, England, 1999).

Thus, by the use of single amino acid substitutions, the binding properties and/or cross-reactivity of peptide ligands for HLA supertype molecules can be modulated.

Example 16 Identification and Confirmation of 158P3D2-Derived Sequences with HLA-DR Binding Motifs

Peptide epitopes bearing an HLA class II supermotif or motif are identified and confirmed as outlined below using methodology similar to that described for HLA Class I peptides.

Selection of HLA-DR-Supermotif-Bearing Epitopes.

To identify 158P3D2-derived, HLA class II HTL epitopes, a 158P3D2 antigen is analyzed for the presence of sequences bearing an HLA-DR-motif or supermotif. Specifically, 15-mer sequences are selected comprising a DR-supermotif, comprising a 9-mer core, and three-residue N- and C-terminal flanking regions (15 amino acids total).

Protocols for predicting peptide binding to DR molecules have been developed (Southwood et al., J. Immunol. 160:3363-3373, 1998). These protocols, specific for individual DR molecules, allow the scoring, and ranking, of 9-mer core regions. Each protocol not only scores peptide sequences for the presence of DR-supermotif primary anchors (i.e., at position 1 and position 6) within a 9-mer core, but additionally evaluates sequences for the presence of secondary anchors. Using allele-specific selection tables (see, e.g., Southwood et al., ibid.), it has been found that these protocols efficiently select peptide sequences with a high probability of binding a particular DR molecule. Additionally, it has been found that performing these protocols in tandem, specifically those for DR1, DR4w4, and DR7, can efficiently select DR cross-reactive peptides.

The 158P3D2-derived peptides identified above are tested for their binding capacity for various common HLA-DR molecules. All peptides are initially tested for binding to the DR molecules in the primary panel: DR1, DR4w4, and DR7. Peptides binding at least two of these three DR molecules are then tested for binding to DR2w2 β1, DR2w2 β2, DR6w19, and DR9 molecules in secondary assays. Finally, peptides binding at least two of the four secondary panel DR molecules, and thus cumulatively at least four of seven different DR molecules, are screened for binding to DR4w15, DR5w11, and DR8w2 molecules in tertiary assays. Peptides binding at least seven of the ten DR molecules comprising the primary, secondary, and tertiary screening assays are considered cross-reactive DR binders. 158P3D2-derived peptides found to bind common HLA-DR alleles are of particular interest.

Selection of DR3 Motif Peptides

Because HLA-DR3 is an allele that is prevalent in Caucasian, Black, and Hispanic populations, DR3 binding capacity is a relevant criterion in the selection of HTL epitopes. Thus, peptides shown to be candidates may also be assayed for their DR3 binding capacity. However, in view of the binding specificity of the DR3 motif, peptides binding only to DR3 can also be considered as candidates for inclusion in a vaccine formulation.

To efficiently identify peptides that bind DR3, target 158P3D2 antigens are analyzed for sequences carrying one of the two DR3-specific binding motifs reported by Geluk et al. (J. Immunol. 152:5742-5748, 1994). The corresponding peptides are then synthesized and confirmed as having the ability to bind DR3 with an affinity of 1 μM or better, i.e., less than 1 μM. Peptides are found that meet this binding criterion and qualify as HLA class II high affinity binders.

DR3 binding epitopes identified in this manner are included in vaccine compositions with DR supermotif-bearing peptide epitopes.

Similarly to the case of HLA class I motif-bearing peptides, the class II motif-bearing peptides are analoged to improve affinity or cross-reactivity. For example, aspartic acid at position 4 of the 9-mer core sequence is an optimal residue for DR3 binding, and substitution for that residue often improves DR 3 binding.

Example 17 Immunogenicity of 158P3D2-Derived HTL Epitopes

This example determines immunogenic DR supermotif- and DR3 motif-bearing epitopes among those identified using the methodology set forth herein.

Immunogenicity of HTL epitopes are confirmed in a manner analogous to the determination of immunogenicity of CTL epitopes, by assessing the ability to stimulate HTL responses and/or by using appropriate transgenic mouse models. Immunogenicity is determined by screening for: 1.) in vitro primary induction using normal PBMC or 2.) recall responses from patients who have 158P3D2-expressing tumors.

Example 18 Calculation of Phenotypic Frequencies of HLA-Supertypes in Various Ethnic Backgrounds to Determine Breadth of Population Coverage

This example illustrates the assessment of the breadth of population coverage of a vaccine composition comprised of multiple epitopes comprising multiple supermotifs and/or motifs.

In order to analyze population coverage, gene frequencies of HLA alleles are determined. Gene frequencies for each HLA allele are calculated from antigen or allele frequencies utilizing the binomial distribution formulae gf=1−(SQRT(1-af)) (see, e.g., Sidney et al., Human Immunol. 45:79-93, 1996). To obtain overall phenotypic frequencies, cumulative gene frequencies are calculated, and the cumulative antigen frequencies derived by the use of the inverse formula [af=1−(1−Cgf)²].

Where frequency data is not available at the level of DNA typing, correspondence to the serologically defined antigen frequencies is assumed. To obtain total potential supertype population coverage no linkage disequilibrium is assumed, and only alleles confirmed to belong to each of the supertypes are included (minimal estimates). Estimates of total potential coverage achieved by inter-loci combinations are made by adding to the A coverage the proportion of the non-A covered population that could be expected to be covered by the B alleles considered (e.g., total=A+B*(1−A)). Confirmed members of the A3-like supertype are A3, A11, A31, A*3301, and A*6801. Although the A3-like supertype may also include A34, A66, and A*7401, these alleles were not included in overall frequency calculations. Likewise, confirmed members of the A2-like supertype family are A*0201, A*0202, A*0203, A*0204, A*0205, A*0206, A*0207, A*6802, and A*6901. Finally, the B7-like supertype-confirmed alleles are: B7, B*3501-03, B51, B*5301, B*5401, B*5501-2, B*5601, B*6701, and B*7801 (potentially also B*1401, B*3504-06, B*4201, and B*5602).

Population coverage achieved by combining the A2-, A3- and B7-supertypes is approximately 86% in five major ethnic groups. Coverage may be extended by including peptides bearing the A1 and A24 motifs. On average, A1 is present in 12% and A24 in 29% of the population across five different major ethnic groups (Caucasian, North American Black, Chinese, Japanese, and Hispanic). Together, these alleles are represented with an average frequency of 39% in these same ethnic populations. The total coverage across the major ethnicities when A1 and A24 are combined with the coverage of the A2-, A3- and B7-supertype alleles is >95%. An analogous approach can be used to estimate population coverage achieved with combinations of class II motif-bearing epitopes.

Immunogenicity studies in humans (e.g., Bertoni et al., J. Clin. Invest. 100:503, 1997; Doolan et al., Immunity 7:97, 1997; and Threlkeld et al., J. Immunol. 159:1648, 1997) have shown that highly cross-reactive binding peptides are almost always recognized as epitopes. The use of highly cross-reactive binding peptides is an important selection criterion in identifying candidate epitopes for inclusion in a vaccine that is immunogenic in a diverse population.

With a sufficient number of epitopes (as disclosed herein and from the art), an average population coverage is predicted to be greater than 95% in each of five major ethnic populations. The game theory Monte Carlo simulation analysis, which is known in the art (see e.g., Osborne, M. J. and Rubinstein, A. “A course in game theory” MIT Press, 1994), can be used to estimate what percentage of the individuals in a population comprised of the Caucasian, North American Black, Japanese, Chinese, and Hispanic ethnic groups would recognize the vaccine epitopes described herein. A preferred percentage is 90%. A more preferred percentage is 95%.

Example 19 CTL Recognition of Endogenously Processed Antigens after Priming

This example confirms that CTL induced by native or analoged peptide epitopes identified and selected as described herein recognize endogenously synthesized, i.e., native antigens.

Effector cells isolated from transgenic mice that are immunized with peptide epitopes, for example HLA-A2 supermotif-bearing epitopes, are re-stimulated in vitro using peptide-coated stimulator cells. Six days later, effector cells are assayed for cytotoxicity and the cell lines that contain peptide-specific cytotoxic activity are further re-stimulated. An additional six days later, these cell lines are tested for cytotoxic activity on ⁵¹Cr labeled Jurkat-A2.1/K^(b) target cells in the absence or presence of peptide, and also tested on ⁵¹Cr labeled target cells bearing the endogenously synthesized antigen, i.e. cells that are stably transfected with 158P3D2 expression vectors.

The results demonstrate that CTL lines obtained from animals primed with peptide epitope recognize endogenously synthesized 158P3D2 antigen. The choice of transgenic mouse model to be used for such an analysis depends upon the epitope(s) that are being evaluated. In addition to HLA-A*0201/K^(b) transgenic mice, several other transgenic mouse models including mice with human A11, which may also be used to evaluate A3 epitopes, and B7 alleles have been characterized and others (e.g., transgenic mice for HLA-A1 and A24) are being developed. HLA-DR1 and HLA-DR3 mouse models have also been developed, which may be used to evaluate HTL epitopes.

Example 20 Activity of CTL-HTL Conjugated Epitopes in Transgenic Mice

This example illustrates the induction of CTLs and HTLs in transgenic mice, by use of a 158P3D2-derived CTL and HTL peptide vaccine compositions. The vaccine composition used herein comprise peptides to be administered to a patient with a 158P3D2-expressing tumor. The peptide composition can comprise multiple CTL and/or HTL epitopes. The epitopes are identified using methodology as described herein. This example also illustrates that enhanced immunogenicity can be achieved by inclusion of one or more HTL epitopes in a CTL vaccine composition; such a peptide composition can comprise an HTL epitope conjugated to a CTL epitope. The CTL epitope can be one that binds to multiple HLA family members at an affinity of 500 nM or less, or analogs of that epitope. The peptides may be lipidated, if desired.

Immunization procedures: Immunization of transgenic mice is performed as described (Alexander et al., J. Immunol. 159:4753-4761, 1997). For example, A2/K^(b) mice, which are transgenic for the human HLA A2.1 allele and are used to confirm the immunogenicity of HLA-A*0201 motif- or HLA-A2 supermotif-bearing epitopes, and are primed subcutaneously (base of the tail) with a 0.1 ml of peptide in Incomplete Freund's Adjuvant, or if the peptide composition is a lipidated CTL/HTL conjugate, in DMSO/saline, or if the peptide composition is a polypeptide, in PBS or Incomplete Freund's Adjuvant. Seven days after priming, splenocytes obtained from these animals are restimulated with syngenic irradiated LPS-activated lymphoblasts coated with peptide.

Cell lines: Target cells for peptide-specific cytotoxicity assays are Jurkat cells transfected with the HLA-A2.1/K^(b) chimeric gene (e.g., Vitiello et al., J. Exp. Med. 173:1007, 1991)

In vitro CTL activation: One week after priming, spleen cells (30×10⁶ cells/flask) are co-cultured at 37° C. with syngeneic, irradiated (3000 rads), peptide coated lymphoblasts (10×10⁶ cells/flask) in 10 ml of culture medium/T25 flask. After six days, effector cells are harvested and assayed for cytotoxic activity.

Assay for cytotoxic activity: Target cells (1.0 to 1.5×10⁶) are incubated at 37° C. in the presence of 200 μl of ⁵¹Cr. After 60 minutes, cells are washed three times and resuspended in R10 medium. Peptide is added where required at a concentration of 1 μg/ml. For the assay, 10⁴ ⁵¹Cr-labeled target cells are added to different concentrations of effector cells (final volume of 200 μl) in U-bottom 96-well plates. After a six hour incubation period at 37° C., a 0.1 ml aliquot of supernatant is removed from each well and radioactivity is determined in a Micromedic automatic gamma counter. The percent specific lysis is determined by the formula: percent specific release=100×(experimental release−spontaneous release)/(maximum release−spontaneous release). To facilitate comparison between separate CTL assays run under the same conditions, % ⁵¹Cr release data is expressed as lytic units/10⁶ cells. One lytic unit is arbitrarily defined as the number of effector cells required to achieve 30% lysis of 10,000 target cells in a six hour ⁵¹Cr release assay. To obtain specific lytic units/10⁶, the lytic units/10⁶ obtained in the absence of peptide is subtracted from the lytic units/10⁶ obtained in the presence of peptide. For example, if 30% ⁵¹Cr release is obtained at the effector (E): target (T) ratio of 50:1 (i.e., 5×10⁵ effector cells for 10,000 targets) in the absence of peptide and 5:1 (i.e., 5×10⁴ effector cells for 10,000 targets) in the presence of peptide, the specific lytic units would be: [(1/50,000)−(1/500,000)]×106=18 LU.

The results are analyzed to assess the magnitude of the CTL responses of animals injected with the immunogenic CTL/HTL conjugate vaccine preparation and are compared to the magnitude of the CTL response achieved using, for example, CTL epitopes as outlined above in Example 14. Analyses similar to this may be performed to confirm the immunogenicity of peptide conjugates containing multiple CTL epitopes and/or multiple HTL epitopes. In accordance with these procedures, it is found that a CTL response is induced, and concomitantly that an HTL response is induced upon administration of such compositions.

Example 21 Selection of CTL and HTL Epitopes for Inclusion in an 158P3D2-Specific Vaccine

This example illustrates a procedure for selecting peptide epitopes for vaccine compositions of the invention. The peptides in the composition can be in the form of a nucleic acid sequence, either single or one or more sequences (i.e., minigene) that encodes peptide(s), or can be single and/or polyepitopic peptides.

The following principles are utilized when selecting a plurality of epitopes for inclusion in a vaccine composition. Each of the following principles is balanced in order to make the selection.

Epitopes are selected which, upon administration, mimic immune responses that are correlated with 158P3D2 clearance. The number of epitopes used depends on observations of patients who spontaneously clear 158P3D2. For example, if it has been observed that patients who spontaneously clear 158P3D2 generate an immune response to at least three (3) from 158P3D2 antigen, then three or four (3-4) epitopes should be included for HLA class 1. A similar rationale is used to determine HLA class II epitopes.

Epitopes are often selected that have a binding affinity of an IC₅₀ of 500 nM or less for an HLA class I molecule, or for class II, an IC₅₀ of 1000 nM or less; or HLA Class I peptides with high binding scores from the BIMAS web site, at URL bimas.dcrt.nih.gov/.

In order to achieve broad coverage of the vaccine through out a diverse population, sufficient supermotif bearing peptides, or a sufficient array of allele-specific motif bearing peptides, are selected to give broad population coverage. In one embodiment, epitopes are selected to provide at least 80% population coverage. A Monte Carlo analysis, a statistical evaluation known in the art, can be employed to assess breadth, or redundancy, of population coverage.

When creating polyepitopic compositions, or a minigene that encodes same, it is typically desirable to generate the smallest peptide possible that encompasses the epitopes of interest. The principles employed are similar, if not the same, as those employed when selecting a peptide comprising nested epitopes. For example, a protein sequence for the vaccine composition is selected because it has maximal number of epitopes contained within the sequence, i.e., it has a high concentration of epitopes. Epitopes may be nested or overlapping (i.e., frame shifted relative to one another). For example, with overlapping epitopes, two 9-mer epitopes and one 10-mer epitope can be present in a 10 amino acid peptide. Each epitope can be exposed and bound by an HLA molecule upon administration of such a peptide. A multi-epitopic, peptide can be generated synthetically, recombinantly, or via cleavage from the native source. Alternatively, an analog can be made of this native sequence, whereby one or more of the epitopes comprise substitutions that alter the cross-reactivity and/or binding affinity properties of the polyepitopic peptide. Such a vaccine composition is administered for therapeutic or prophylactic purposes. This embodiment provides for the possibility that an as yet undiscovered aspect of immune system processing will apply to the native nested sequence and thereby facilitate the production of therapeutic or prophylactic immune response-inducing vaccine compositions. Additionally such an embodiment provides for the possibility of motif-bearing epitopes for an HLA makeup that is presently unknown. Furthermore, this embodiment (absent the creating of any analogs) directs the immune response to multiple peptide sequences that are actually present in 158P3D2, thus avoiding the need to evaluate any junctional epitopes. Lastly, the embodiment provides an economy of scale when producing nucleic acid vaccine compositions. Related to this embodiment, computer programs can be derived in accordance with principles in the art, which identify in a target sequence, the greatest number of epitopes per sequence length.

A vaccine composition comprised of selected peptides, when administered, is safe, efficacious, and elicits an immune response similar in magnitude to an immune response that controls or clears cells that bear or overexpress 158P3D2.

Example 22 Construction of “Minigene” Multi-Epitope DNA Plasmids

This example discusses the construction of a minigene expression plasmid. Minigene plasmids may, of course, contain various configurations of B cell, CTL and/or HTL epitopes or epitope analogs as described herein.

A minigene expression plasmid typically includes multiple CTL and HTL peptide epitopes. In the present example, HLA-A2, -A3, -B7 supermotif-bearing peptide epitopes and HLA-A1 and -A24 motif-bearing peptide epitopes are used in conjunction with DR supermotif-bearing epitopes and/or DR3 epitopes. HLA class I supermotif or motif-bearing peptide epitopes derived 158P3D2, are selected such that multiple supermotifs/motifs are represented to ensure broad population coverage. Similarly, HLA class II epitopes are selected from 158P3D2 to provide broad population coverage, i.e. both HLA DR-1-4-7 supermotif-bearing epitopes and HLA DR-3 motif-bearing epitopes are selected for inclusion in the minigene construct. The selected CTL and HTL epitopes are then incorporated into a minigene for expression in an expression vector.

Such a construct may additionally include sequences that direct the HTL epitopes to the endoplasmic reticulum. For example, the Ii protein may be fused to one or more HTL epitopes as described in the art, wherein the CLIP sequence of the Ii protein is removed and replaced with an HLA class II epitope sequence so that HLA class II epitope is directed to the endoplasmic reticulum, where the epitope binds to an HLA class II molecules.

This example illustrates the methods to be used for construction of a minigene-bearing expression plasmid. Other expression vectors that may be used for minigene compositions are available and known to those of skill in the art.

The minigene DNA plasmid of this example contains a consensus Kozak sequence and a consensus murine kappa Ig-light chain signal sequence followed by CTL and/or HTL epitopes selected in accordance with principles disclosed herein. The sequence encodes an open reading frame fused to the Myc and His antibody epitope tag coded for by the pcDNA 3.1 Myc-His vector.

Overlapping oligonucleotides that can, for example, average about 70 nucleotides in length with 15 nucleotide overlaps, are synthesized and HPLC-purified. The oligonucleotides encode the selected peptide epitopes as well as appropriate linker nucleotides, Kozak sequence, and signal sequence. The final multiepitope minigene is assembled by extending the overlapping oligonucleotides in three sets of reactions using PCR. A Perkin/Elmer 9600 PCR machine is used and a total of 30 cycles are performed using the following conditions: 95° C. for 15 sec, annealing temperature (5° below the lowest calculated Tm of each primer pair) for 30 sec, and 72° C. for 1 min.

For example, a minigene is prepared as follows. For a first PCR reaction, 5 μg of each of two oligonucleotides are annealed and extended: In an example using eight oligonucleotides, i.e., four pairs of primers, oligonucleotides 1+2, 3+4, 5+6, and 7+8 are combined in 100 μl reactions containing Pfu polymerase buffer (1×=10 mM KCL, 10 mM (NH4)₂SO₄, 20 mM Tris-chloride, pH 8.75, 2 mM MgSO₄, 0.1% Triton X-100, 100 μg/ml BSA), 0.25 mM each dNTP, and 2.5 U of Pfu polymerase. The full-length dimer products are gel-purified, and two reactions containing the product of 1+2 and 3+4, and the product of 5+6 and 7+8 are mixed, annealed, and extended for 10 cycles. Half of the two reactions are then mixed, and 5 cycles of annealing and extension carried out before flanking primers are added to amplify the full length product. The full-length product is gel-purified and cloned into pCR-blunt (Invitrogen) and individual clones are screened by sequencing.

Example 23 The Plasmid Construct and the Degree to which it Induces Immunogenicity

The degree to which a plasmid construct, for example a plasmid constructed in accordance with the previous Example, is able to induce immunogenicity is confirmed in vitro by determining epitope presentation by APC following transduction or transfection of the APC with an epitope-expressing nucleic acid construct. Such a study determines “antigenicity” and allows the use of human APC. The assay determines the ability of the epitope to be presented by the APC in a context that is recognized by a T cell by quantifying the density of epitope-HLA class I complexes on the cell surface. Quantitation can be performed by directly measuring the amount of peptide eluted from the APC (see, e.g., Sijts et al., J. Immunol. 156:683-692, 1996; Demotz et al., Nature 342:682-684, 1989); or the number of peptide-HLA class I complexes can be estimated by measuring the amount of lysis or lymphokine release induced by diseased or transfected target cells, and then determining the concentration of peptide necessary to obtain equivalent levels of lysis or lymphokine release (see, e.g., Kageyama et al., J. Immunol. 154:567-576, 1995).

Alternatively, immunogenicity is confirmed through in vivo injections into mice and subsequent in vitro assessment of CTL and HTL activity, which are analyzed using cytotoxicity and proliferation assays, respectively, as detailed e.g., in Alexander et al., Immunity 1:751-761, 1994.

For example, to confirm the capacity of a DNA minigene construct containing at least one HLA-A2 supermotif peptide to induce CTLs in vivo, HLA-A2.11 K^(b) transgenic mice, for example, are immunized intramuscularly with 100 μg of naked cDNA. As a means of comparing the level of CTLs induced by cDNA immunization, a control group of animals is also immunized with an actual peptide composition that comprises multiple epitopes synthesized as a single polypeptide as they would be encoded by the minigene.

Splenocytes from immunized animals are stimulated twice with each of the respective compositions

(peptide epitopes encoded in the minigene or the polyepitopic peptide), then assayed for peptide-specific cytotoxic activity in a ⁵¹Cr release assay. The results indicate the magnitude of the CTL response directed against the A2-restricted epitope, thus indicating the in vivo immunogenicity of the minigene vaccine and polyepitopic vaccine.

It is, therefore, found that the minigene elicits immune responses directed toward the HLA-A2 supermotif peptide epitopes as does the polyepitopic peptide vaccine. A similar analysis is also performed using other HLA-A3 and HLA-B7 transgenic mouse models to assess CTL induction by HLA-A3 and HLA-B7 motif or supermotif epitopes, whereby it is also found that the minigene elicits appropriate immune responses directed toward the provided epitopes.

To confirm the capacity of a class II epitope-encoding minigene to induce HTLs in vivo, DR transgenic mice, or for those epitopes that cross react with the appropriate mouse MHC molecule, I-A^(b)-restricted mice, for example, are immunized intramuscularly with 100 μg of plasmid DNA. As a means of comparing the level of HTLs induced by DNA immunization, a group of control animals is also immunized with an actual peptide composition emulsified in complete Freund's adjuvant. CD4+ T cells, i.e. HTLs, are purified from splenocytes of immunized animals and stimulated with each of the respective compositions (peptides encoded in the minigene). The HTL response is measured using a ³H-thymidine incorporation proliferation assay, (see, e.g., Alexander et al. Immunity 1:751-761, 1994). The results indicate the magnitude of the HTL response, thus demonstrating the in vivo immunogenicity of the minigene.

DNA minigenes, constructed as described in the previous Example, can also be confirmed as a vaccine in combination with a boosting agent using a prime boost protocol. The boosting agent can consist of recombinant protein (e.g., Barnett et al., Aids Res. and Human Retroviruses 14, Supplement 3:S299-S309, 1998) or recombinant vaccinia, for example, expressing a minigene or DNA encoding the complete protein of interest (see, e.g., Hanke et al., Vaccine 16:439-445, 1998; Sedegah et al., Proc. Natl. Acad Sci USA 95:7648-53, 1998; Hanke and McMichael, Immunol. Letters 66:177-181, 1999; and Robinson et al., Nature Med. 5:526-34, 1999).

For example, the efficacy of the DNA minigene used in a prime boost protocol is initially evaluated in transgenic mice. In this example, A2.1/K^(b) transgenic mice are immunized IM with 100 μg of a DNA minigene encoding the immunogenic peptides including at least one HLA-A2 supermotif-bearing peptide. After an incubation period (ranging from 3-9 weeks), the mice are boosted IP with 10⁷ pfu/mouse of a recombinant vaccinia virus expressing the same sequence encoded by the DNA minigene. Control mice are immunized with 100 μg of DNA or recombinant vaccinia without the minigene sequence, or with DNA encoding the minigene, but without the vaccinia boost. After an additional incubation period of two weeks, splenocytes from the mice are immediately assayed for peptide-specific activity in an ELISPOT assay. Additionally, splenocytes are stimulated in vitro with the A2-restricted peptide epitopes encoded in the minigene and recombinant vaccinia, then assayed for peptide-specific activity in an alpha, beta and/or gamma IFN ELISA.

It is found that the minigene utilized in a prime-boost protocol elicits greater immune responses toward the HLA-A2 supermotif peptides than with DNA alone. Such an analysis can also be performed using HLA-A 11 or HLA-B7 transgenic mouse models to assess CTL induction by HLA-A3 or HLA-B7 motif or supermotif epitopes. The use of prime boost protocols in humans is described below in Example 31.

Example 24 Peptide Compositions for Prophylactic Uses

Vaccine compositions of the present invention can be used to prevent 158P3D2 expression in persons who are at risk for tumors that bear this antigen. For example, a polyepitopic peptide epitope composition (or a nucleic acid comprising the same) containing multiple CTL and HTL epitopes such as those selected in the above Examples, which are also selected to target greater than 80% of the population, is administered to individuals at risk for a 158P3D2-associated tumor.

For example, a peptide-based composition is provided as a single polypeptide that encompasses multiple epitopes. The vaccine is typically administered in a physiological solution that comprises an adjuvant, such as Incomplete Freunds Adjuvant. The dose of peptide for the initial immunization is from about 1 to about 50,000 μg, generally 100-5,000 μg, for a 70 kg patient. The initial administration of vaccine is followed by booster dosages at 4 weeks followed by evaluation of the magnitude of the immune response in the patient, by techniques that determine the presence of epitope-specific CTL populations in a PBMC sample. Additional booster doses are administered as required. The composition is found to be both safe and efficacious as a prophylaxis against 158P3D2-associated disease.

Alternatively, a composition typically comprising transfecting agents is used for the administration of a nucleic acid-based vaccine in accordance with methodologies known in the art and disclosed herein.

Example 25 Polyepitopic Vaccine Compositions Derived from Native 158P3D2 Sequences

A native 158P3D2 polyprotein sequence is analyzed, preferably using computer algorithms defined for each class I and/or class II supermotif or motif, to identify “relatively short” regions of the polyprotein that comprise multiple epitopes. The “relatively short” regions are preferably less in length than an entire native antigen. This relatively short sequence that contains multiple distinct or overlapping, “nested” epitopes is selected; it can be used to generate a minigene construct. The construct is engineered to express the peptide, which corresponds to the native protein sequence. The “relatively short” peptide is generally less than 250 amino acids in length, often less than 100 amino acids in length, preferably less than 75 amino acids in length, and more preferably less than 50 amino acids in length. The protein sequence of the vaccine composition is selected because it has maximal number of epitopes contained within the sequence, i.e., it has a high concentration of epitopes. As noted herein, epitope motifs may be nested or overlapping (i.e., frame shifted relative to one another). For example, with overlapping epitopes, two 9-mer epitopes and one 10-mer epitope can be present in a 10 amino acid peptide. Such a vaccine composition is administered for therapeutic or prophylactic purposes.

The vaccine composition will include, for example, multiple CTL epitopes from 158P3D2 antigen and at least one HTL epitope. This polyepitopic native sequence is administered either as a peptide or as a nucleic acid sequence which encodes the peptide. Alternatively, an analog can be made of this native sequence, whereby one or more of the epitopes comprise substitutions that alter the cross-reactivity and/or binding affinity properties of the polyepitopic peptide.

The embodiment of this example provides for the possibility that an as yet undiscovered aspect of immune system processing will apply to the native nested sequence and thereby facilitate the production of therapeutic or prophylactic immune response-inducing vaccine compositions. Additionally such an embodiment provides for the possibility of motif-bearing epitopes for an HLA makeup that is presently unknown. Furthermore, this embodiment (excluding an analoged embodiment) directs the immune response to multiple peptide sequences that are actually present in native 158P3D2, thus avoiding the need to evaluate any junctional epitopes. Lastly, the embodiment provides an economy of scale when producing peptide or nucleic acid vaccine compositions.

Related to this embodiment, computer programs are available in the art which can be used to identify in a target sequence, the greatest number of epitopes per sequence length.

Example 26 Polyepitopic Vaccine Compositions from Multiple Antigens

The 158P3D2 peptide epitopes of the present invention are used in conjunction with epitopes from other target tumor-associated antigens, to create a vaccine composition that is useful for the prevention or treatment of cancer that expresses 158P3D2 and such other antigens. For example, a vaccine composition can be provided as a single polypeptide that incorporates multiple epitopes from 158P3D2 as well as tumor-associated antigens that are often expressed with a target cancer associated with 158P3D2 expression, or can be administered as a composition comprising a cocktail of one or more discrete epitopes. Alternatively, the vaccine can be administered as a minigene construct or as dendritic cells which have been loaded with the peptide epitopes in vitro.

Example 27 Use of Peptides to Evaluate an Immune Response

Peptides of the invention may be used to analyze an immune response for the presence of specific antibodies, CTL or HTL directed to 158P3D2. Such an analysis can be performed in a manner described by Ogg et al., Science 279:2103-2106, 1998. In this Example, peptides in accordance with the invention are used as a reagent for diagnostic or prognostic purposes, not as an immunogen.

In this example highly sensitive human leukocyte antigen tetrameric complexes (“tetramers”) are used for a cross-sectional analysis of, for example, 158P3D2 HLA-A*0201-specific CTL frequencies from HLA A*0201-positive individuals at different stages of disease or following immunization comprising an 158P3D2 peptide containing an A*0201 motif. Tetrameric complexes are synthesized as described (Musey et al., N. Engl. J. Med. 337:1267, 1997). Briefly, purified HLA heavy chain (A*0201 in this example) and β2-microglobulin are synthesized by means of a prokaryotic expression system. The heavy chain is modified by deletion of the transmembrane-cytosolic tail and COOH-terminal addition of a sequence containing a BirA enzymatic biotinylation site. The heavy chain, P2-microglobulin, and peptide are refolded by dilution. The 45-kD refolded product is isolated by fast protein liquid chromatography and then biotinylated by BirA in the presence of biotin (Sigma, St. Louis, Mo.), adenosine 5′ triphosphate and magnesium. Streptavidin-phycoerythrin conjugate is added in a 1:4 molar ratio, and the tetrameric product is concentrated to 1 mg/ml. The resulting product is referred to as tetramer-phycoerythrin.

For the analysis of patient blood samples, approximately one million PBMCs are centrifuged at 300 g for 5 minutes and resuspended in 50 μl of cold phosphate-buffered saline. Tri-color analysis is performed with the tetramer-phycoerythrin, along with anti-CD8-Tricolor, and anti-CD38. The PBMCs are incubated with tetramer and antibodies on ice for 30 to 60 min and then washed twice before formaldehyde fixation. Gates are applied to contain >99.98% of control samples. Controls for the tetramers include both A*0201-negative individuals and A*0201-positive non-diseased donors. The percentage of cells stained with the tetramer is then determined by flow cytometry. The results indicate the number of cells in the PBMC sample that contain epitope-restricted CTLs, thereby readily indicating the extent of immune response to the 158P3D2 epitope, and thus the status of exposure to 158P3D2, or exposure to a vaccine that elicits a protective or therapeutic response.

Example 28 Use of Peptide Epitopes to Evaluate Recall Responses

The peptide epitopes of the invention are used as reagents to evaluate T cell responses, such as acute or recall responses, in patients. Such an analysis may be performed on patients who have recovered from 158P3D2-associated disease or who have been vaccinated with an 158P3D2 vaccine.

For example, the class I restricted CTL response of persons who have been vaccinated may be analyzed. The vaccine may be any 158P3D2 vaccine. PBMC are collected from vaccinated individuals and HLA typed. Appropriate peptide epitopes of the invention that, optimally, bear supermotifs to provide cross-reactivity with multiple HLA supertype family members, are then used for analysis of samples derived from individuals who bear that HLA type.

PBMC from vaccinated individuals are separated on Ficoll-Histopaque density gradients (Sigma Chemical Co., St. Louis, Mo.), washed three times in HBSS (GIBCO Laboratories), resuspended in RPMI-1640 (GIBCO Laboratories) supplemented with L-glutamine (2 mM), penicillin (50 U/ml), streptomycin (50 μg/ml), and Hepes (10 mM) containing 10% heat-inactivated human AB serum (complete RPMI) and plated using microculture formats. A synthetic peptide comprising an epitope of the invention is added at 10 μg/ml to each well and HBV core 128-140 epitope is added at 1 μg/ml to each well as a source of T cell help during the first week of stimulation.

In the microculture format, 4×10⁵ PBMC are stimulated with peptide in 8 replicate cultures in 96-well round bottom plate in 100 μl/well of complete RPMI. On days 3 and 10, 100 μl of complete RPMI and 20 U/ml final concentration of rIL-2 are added to each well. On day 7 the cultures are transferred into a 96-well flat-bottom plate and restimulated with peptide, rIL-2 and 10⁵ irradiated (3,000 rad) autologous feeder cells. The cultures are tested for cytotoxic activity on day 14. A positive CTL response requires two or more of the eight replicate cultures to display greater than 10% specific ⁵¹Cr release, based on comparison with non-diseased control subjects as previously described (Rehermann, et al., Nature Med. 2:1104, 1108, 1996; Rehermann et al., J. Clin. Invest. 97:1655-1665, 1996; and Rehermann et al. J. Clin. Invest. 98:1432-1440, 1996).

Target cell lines are autologous and allogeneic EBV-transformed B-LCL that are either purchased from the American Society for Histocompatibility and Immunogenetics (ASHI, Boston, Mass.) or established from the pool of patients as described (Guilhot, et al. J. Virol. 66:2670-2678, 1992).

Cytotoxicity assays are performed in the following manner. Target cells consist of either allogeneic HLA-matched or autologous EBV-transformed B lymphoblastoid cell line that are incubated overnight with the synthetic peptide epitope of the invention at 10 μM, and labeled with 100 μCi of ⁵¹Cr (Amersham Corp., Arlington Heights, Ill.) for 1 hour after which they are washed four times with HBSS.

Cytolytic activity is determined in a standard 4-h, split well ⁵¹Cr release assay using U-bottomed 96 well plates containing 3,000 targets/well. Stimulated PBMC are tested at effector/target (E/T) ratios of 20-50:1 on day 14. Percent cytotoxicity is determined from the formula: 100×[(experimental release-spontaneous release)/maximum release-spontaneous release)]. Maximum release is determined by lysis of targets by detergent (2% Triton X-100; Sigma Chemical Co., St. Louis, Mo.). Spontaneous release is <25% of maximum release for all experiments.

The results of such an analysis indicate the extent to which HLA-restricted CTL populations have been stimulated by previous exposure to 158P3D2 or an 158P3D2 vaccine.

Similarly, Class II restricted HTL responses may also be analyzed. Purified PBMC are cultured in a 96-well flat bottom plate at a density of 1.5×10⁵ cells/well and are stimulated with 10 μg/ml synthetic peptide of the invention, whole 158P3D2 antigen, or PHA. Cells are routinely plated in replicates of 4-6 wells for each condition. After seven days of culture, the medium is removed and replaced with fresh medium containing 10 U/ml IL-2. Two days later, 1 μCi ³H-thymidine is added to each well and incubation is continued for an additional 18 hours. Cellular DNA is then harvested on glass fiber mats and analyzed for 3H-thymidine incorporation. Antigen-specific T cell proliferation is calculated as the ratio of ³H-thymidine incorporation in the presence of antigen divided by the ³H-thymidine incorporation in the absence of antigen.

Example 29 Induction of Specific Ctl Response in Humans

A human clinical trial for an immunogenic composition comprising CTL and HTL epitopes of the invention is set up as an IND Phase I, dose escalation study and carried out as a randomized, double-blind, placebo-controlled trial. Such a trial is designed, for example, as follows:

A total of about 27 individuals are enrolled and divided into 3 groups:

Group I: 3 subjects are injected with placebo and 6 subjects are injected with 5 μg of peptide composition;

Group II: 3 subjects are injected with placebo and 6 subjects are injected with 50 μg peptide composition;

Group III: 3 subjects are injected with placebo and 6 subjects are injected with 500 μg of peptide composition.

After 4 weeks following the first injection, all subjects receive a booster inoculation at the same dosage.

The endpoints measured in this study relate to the safety and tolerability of the peptide composition as well as its immunogenicity. Cellular immune responses to the peptide composition are an index of the intrinsic activity of this the peptide composition, and can therefore be viewed as a measure of biological efficacy. The following summarize the clinical and laboratory data that relate to safety and efficacy endpoints.

Safety: The incidence of adverse events is monitored in the placebo and drug treatment group and assessed in terms of degree and reversibility.

Evaluation of Vaccine Efficacy: For evaluation of vaccine efficacy, subjects are bled before and after injection. Peripheral blood mononuclear cells are isolated from fresh heparinized blood by Ficoll-Hypaque density gradient centrifugation, aliquoted in freezing media and stored frozen. Samples are assayed for CTL and HTL activity.

The vaccine is found to be both safe and efficacious.

Example 30 Phase II Trials in Patients Expressing 158P3D2

Phase II trials are performed to study the effect of administering the CTL-HTL peptide compositions to patients having cancer that expresses 158P3D2. The main objectives of the trial are to determine an effective dose and regimen for inducing CTLs in cancer patients that express 158P3D2, to establish the safety of inducing a CTL and HTL response in these patients, and to see to what extent activation of CTLs improves the clinical picture of these patients, as manifested, e.g., by the reduction and/or shrinking of lesions. Such a study is designed, for example, as follows:

The studies are performed in multiple centers. The trial design is an open-label, uncontrolled, dose escalation protocol wherein the peptide composition is administered as a single dose followed six weeks later by a single booster shot of the same dose. The dosages are 50, 500 and 5,000 micrograms per injection. Drug-associated adverse effects (severity and reversibility) are recorded.

There are three patient groupings. The first group is injected with 50 micrograms of the peptide composition and the second and third groups with 500 and 5,000 micrograms of peptide composition, respectively. The patients within each group range in age from 21-65 and represent diverse ethnic backgrounds. All of them have a tumor that expresses 158P3D2.

Clinical manifestations or antigen-specific T-cell responses are monitored to assess the effects of administering the peptide compositions. The vaccine composition is found to be both safe and efficacious in the treatment of 158P3D2-associated disease.

Example 31 Induction of CTL Responses Using a Prime Boost Protocol

A prime boost protocol similar in its underlying principle to that used to confirm the efficacy of a DNA vaccine in transgenic mice, such as described above in Example 23, can also be used for the administration of the vaccine to humans. Such a vaccine regimen can include an initial administration of, for example, naked DNA followed by a boost using recombinant virus encoding the vaccine, or recombinant protein/polypeptide or a peptide mixture administered in an adjuvant.

For example, the initial immunization may be performed using an expression vector, such as that constructed in Example 22 in the form of naked nucleic acid administered IM (or SC or ID) in the amounts of 0.5-5 mg at multiple sites. The nucleic acid (0.1 to 1000 ug) can also be administered using a gene gun. Following an incubation period of 34 weeks, a booster dose is then administered. The booster can be recombinant fowlpox virus administered at a dose of 5-10⁷ to 5×10⁹ pfu. An alternative recombinant virus, such as an MVA, canarypox, adenovirus, or adeno-associated virus, can also be used for the booster, or the polyepitopic protein or a mixture of the peptides can be administered. For evaluation of vaccine efficacy, patient blood samples are obtained before immunization as well as at intervals following administration of the initial vaccine and booster doses of the vaccine. Peripheral blood mononuclear cells are isolated from fresh heparinized blood by Ficoll-Hypaque density gradient centrifugation, aliquoted in freezing media and stored frozen. Samples are assayed for CTL and HTL activity.

Analysis of the results indicates that a magnitude of response sufficient to achieve a therapeutic or protective immunity against 158P3D2 is generated.

Example 32 Administration of Vaccine Compositions Using Dendritic Cells (DC)

Vaccines comprising peptide epitopes of the invention can be administered using APCs, or “professional” APCs such as DC. In this example, peptide-pulsed DC are administered to a patient to stimulate a CTL response in vivo. In this method, dendritic cells are isolated, expanded, and pulsed with a vaccine comprising peptide CTL and HTL epitopes of the invention. The dendritic cells are infused back into the patient to elicit CTL and HTL responses in vivo. The induced CTL and HTL then destroy or facilitate destruction, respectively, of the target cells that bear the 158P3D2 protein from which the epitopes in the vaccine are derived.

For example, a cocktail of epitope-comprising peptides is administered ex vivo to PBMC, or isolated DC therefrom. A pharmaceutical to facilitate harvesting of DC can be used, such as Progenipoietin™ (Monsanto, St. Louis, Mo.) or GM-CSF/IL-4. After pulsing the DC with peptides, and prior to reinfusion into patients, the DC are washed to remove unbound peptides.

As appreciated clinically, and readily determined by one of skill based on clinical outcomes, the number of DC reinfused into the patient can vary (see, e.g., Nature Med 4:328, 1998; Nature Med. 2:52, 1996 and Prostate 32:272, 1997). Although 2−50×10⁶ DC per patient are typically administered, larger number of DC, such as 107 or 108 can also be provided. Such cell populations typically contain between 50-90% DC.

In some embodiments, peptide-loaded PBMC are injected into patients without purification of the DC. For example, PBMC generated after treatment with an agent such as Progenipoietin™ are injected into patients without purification of the DC. The total number of PBMC that are administered often ranges from 10⁸ to 10¹⁰. Generally, the cell doses injected into patients is based on the percentage of DC in the blood of each patient, as determined, for example, by immunofluorescence analysis with specific anti-DC antibodies. Thus, for example, if Progenipoietin™ mobilizes 2% DC in the peripheral blood of a given patient, and that patient is to receive 5×10⁶ DC, then the patient will be injected with a total of 2.5×10⁸ peptide-loaded PBMC. The percent DC mobilized by an agent such as Progenipoietin™ is typically estimated to be between 2-10%, but can vary as appreciated by one of skill in the art.

Ex Vivo Activation of CTL/HTL Responses

Alternatively, ex vivo CTL or HTL responses to 158P3D2 antigens can be induced by incubating, in tissue culture, the patient's, or genetically compatible, CTL or HTL precursor cells together with a source of APC, such as DC, and immunogenic peptides. After an appropriate incubation time (typically about 7-28 days), in which the precursor cells are activated and expanded into effector cells, the cells are infused into the patient, where they will destroy (CTL) or facilitate destruction (HTL) of their specific target cells, i.e., tumor cells.

Example 33 An Alternative Method of Identifying and Confirming Motif-Bearing Peptides

Another method of identifying and confirming motif-bearing peptides is to elute them from cells bearing defined MHC molecules. For example, EBV transformed B cell lines used for tissue typing have been extensively characterized to determine which HLA molecules they express. In certain cases these cells express only a single type of HLA molecule. These cells can be transfected with nucleic acids that express the antigen of interest, e.g. 158P3D2. Peptides produced by endogenous antigen processing of peptides produced as a result of transfection will then bind to HLA molecules within the cell and be transported and displayed on the cell's surface. Peptides are then eluted from the HLA molecules by exposure to mild acid conditions and their amino acid sequence determined, e.g., by mass spectral analysis (e.g., Kubo et al., J. Immunol. 152:3913, 1994). Because the majority of peptides that bind a particular HLA molecule are motif-bearing, this is an alternative modality for obtaining the motif-bearing peptides correlated with the particular HLA molecule expressed on the cell.

Alternatively, cell lines that do not express endogenous HLA molecules can be transfected with an expression construct encoding a single HLA allele. These cells can then be used as described, i.e., they can then be transfected with nucleic acids that encode 158P3D2 to isolate peptides corresponding to 158P3D2 that have been presented on the cell surface. Peptides obtained from such an analysis will bear motif(s) that correspond to binding to the single HLA allele that is expressed in the cell.

As appreciated by one in the art, one can perform a similar analysis on a cell bearing more than one HLA allele and subsequently determine peptides specific for each HLA allele expressed. Moreover, one of skill would also recognize that means other than transfection, such as loading with a protein antigen, can be used to provide a source of antigen to the cell.

Example 34 Complementary Polynucleotides

Sequences complementary to the 158P3D2-encoding sequences, or any parts thereof, are used to detect, decrease, or inhibit expression of naturally occurring 158P3D2. Although use of oligonucleotides comprising from about 15 to 30 base pairs is described, essentially the same procedure is used with smaller or with larger sequence fragments. Appropriate oligonucleotides are designed using, e.g., OLIGO 4.06 software (National Biosciences) and the coding sequence of 158P3D2. To inhibit transcription, a complementary oligonucleotide is designed from the most unique 5′ sequence and used to prevent promoter binding to the coding sequence. To inhibit translation, a complementary oligonucleotide is designed to prevent ribosomal binding to a 158P3D2-encoding transcript.

Example 35 Purification of Naturally-Occurring or Recombinant 158P3D2 Using 158P3D2 Specific Antibodies

Naturally occurring or recombinant 158P3D2 is substantially purified by immunoaffinity chromatography using antibodies specific for 158P3D2. An immunoaffinity column is constructed by covalently coupling anti-158P3D2 antibody to an activated chromatographic resin, such as CNBr-activated SEPHAROSE (Amersham Pharmacia Biotech). After the coupling, the resin is blocked and washed according to the manufacturer's instructions.

Media containing 158P3D2 are passed over the immunoaffinity column, and the column is washed under conditions that allow the preferential absorbance of 158P3D2 (e.g., high ionic strength buffers in the presence of detergent). The column is eluted under conditions that disrupt antibody/158P3D2 binding (e.g., a buffer of pH 2 to pH 3, or a high concentration of a chaotrope, such as urea or thiocyanate ion), and GCR.P is collected.

Example 36 Identification of Molecules Which Interact with 158P3D2

158P3D2, or biologically active fragments thereof, are labeled with 121 l Bolton-Hunter reagent. (See, e.g., Bolton et al. (1973) Biochem. J. 133:529.) Candidate molecules previously arrayed in the wells of a multi-well plate are incubated with the labeled 158P3D2, washed, and any wells with labeled 158P3D2 complex are assayed. Data obtained using different concentrations of 158P3D2 are used to calculate values for the number, affinity, and association of 158P3D2 with the candidate molecules.

Example 37 In Vivo Assay for 158P3D2 Tumor Growth Promotion

The effect of the 158P3D2 protein on tumor cell growth is evaluated in vivo by gene overexpression in tumor-bearing mice. For example, SCID mice are injected subcutaneously on each flank with 1×10⁶ of either NIH-3T3 cells, bladder cancer lines (UM-UC3, J82 or SCABER) and kidney cancer cells (CaKi1, 769-P) containing tkNeo empty vector or 158P3D2. At least two strategies may be used: (1) Constitutive 158P3D2 expression under regulation of a promoter such as a constitutive promoter obtained from the genomes of viruses such as polyoma virus, fowlpox virus (UK 2,211,504 published 5 Jul. 1989), adenovirus (such as Adenovirus 2), bovine papilloma virus, avian sarcoma virus, cytomegalovirus, a retrovirus, hepatitis-B virus and Simian Virus 40 (SV40), or from heterologous mammalian promoters, e.g., the actin promoter or an immunoglobulin promoter, provided such promoters are compatible with the host cell systems, and (2) Regulated expression under control of an inducible vector system, such as ecdysone, tet, etc., provided such promoters are compatible with the host cell systems. Tumor volume is then monitored at the appearance of palpable tumors and followed over time to determine if 158P3D2-expressing cells grow at a faster rate and whether tumors produced by 158P3D2-expressing cells demonstrate characteristics of altered aggressiveness (e.g. enhanced metastasis, vascularization, reduced responsiveness to chemotherapeutic drugs).

Additionally, mice can be implanted with 1×10⁵ of the same cells orthotopically to determine if 158P3D2 has an effect on local growth in the prostate or on the ability of the cells to metastasize, specifically to lungs, lymph nodes, and bone marrow.

The assay is also useful to determine the 158P3D2 inhibitory effect of candidate therapeutic compositions, such as for example, 158P3D2 intrabodies, 158P3D2 antisense molecules and ribozymes.

Example 38 158P3D2 Monoclonal Antibody-Mediated Inhibition of Bladder Tumors In Vivo

The significant expression of 158P3D2 in cancer tissues, its restrictive expression in normal tissues together with its expected cell surface expression makes 158P3D2 an excellent target for antibody therapy. Similarly, 158P3D2 is a target for T cell-based immunotherapy. Thus, the therapeutic efficacy of anti-158P3D2 mAbs in human bladder cancer xenograft mouse models is evaluated by using recombinant cell lines UM-UC3-158P3D2 and J28-158P3D2. Similarly, anti-158P3D2 mAbs are evaluated in human kidney cancer xenograft models such as AGS-K3 and AGS-K6 and in recombinant kidney cell lines such as Caki-158P3D2.

Antibody efficacy on tumor growth and metastasis formation is studied, e.g., in a mouse orthotopic bladder cancer xenograft models and mouse kidney xenograft models. The antibodies can be unconjugated, as discussed in this Example, or can be conjugated to a therapeutic modality, as appreciated in the art. Anti-158P3D2 mAbs inhibit formation of both Caki-158P3D2 and UMUC3-158P3D2 tumor xenografts. Anti-158P3D2 mAbs also retard the growth of established orthotopic tumors and prolonged survival of tumor-bearing mice. These results indicate the utility of anti-158P3D2 mAbs in the treatment of local and advanced stages of kidney and bladder cancer. (See, e.g., (Saffran, D., et al., PNAS 10:1073-1078). These results indicate the use of anti-158P3D2 mAbs in the treatment of bladder and kidney cancer.

Administration of the anti-158P3D2 mAbs led to retardation of established orthotopic tumor growth and inhibition of metastasis to distant sites, resulting in a significant prolongation in the survival of tumor-bearing mice. These studies indicate that 158P3D2 as an attractive target for immunotherapy and demonstrate the therapeutic potential of anti-158P3D2 mAbs for the treatment of local and metastatic prostate cancer. This example demonstrates that unconjugated 158P3D2 monoclonal antibodies are effective to inhibit the growth of human bladder tumor xenografts and human kidney xenografts grown in SCID mice; accordingly a combination of such efficacious monoclonal antibodies is also effective.

Tumor Inhibition Using Multiple Unconjugated 158P3D2 mAbs

Materials and Methods

158P3D2 Monoclonal Antibodies:

Monoclonal antibodies are raised against 158P3D2 as described in Example 11. The antibodies are characterized by ELISA, Western blot, FACS, and immunoprecipitation for their capacity to bind 158P3D2. Epitope mapping data for the anti-158P3D2 mAbs, as determined by ELISA and Western analysis, recognize epitopes on the 158P3D2 protein. Immunohistochemical analysis of prostate cancer tissues and cells with these antibodies is performed.

The monoclonal antibodies are purified from ascites or hybridoma tissue culture supernatants by Protein-G Sepharose chromatography, dialyzed against PBS, filter sterilized, and stored at −20° C. Protein determinations are performed by a Bradford assay (Bio-Rad, Hercules, Calif.). A therapeutic monoclonal antibody or a cocktail comprising a mixture of individual monoclonal antibodies is prepared and used for the treatment of mice receiving subcutaneous or orthotopic injections of LAPC-9 prostate tumor xenografts.

Cancer Xenografts and Cell Lines

Human cancer xenograft models, such as bladder and kidney cancer models, as well as ICR-severe combined immunodeficient (SCID) mice injected with human cell lines expressing or lacking 158P3D2 are used to confirm the role of 158P3D2 in tumor growth and progression. The bladder xenograft is passaged in 6- to 8-week-old male ICR-severe combined immunodeficient (SCID) mice (Taconic Farms) by s.c. trocar implant (Craft, N., et al., supra). The AGS-K3 and AGS-K6 kidney xenografts are also passaged by subcutaneous implants in 6- to 8-week old SCID mice. Single-cell suspensions of tumor cells are prepared as described in Craft, et al. The bladder and kidney carcinoma cell lines UM-UC3, SCABER, J82, 769-P and CaKi (American Type Culture Collection) are maintained in DMEM supplemented with L-glutamine and 10% FBS.

A UMUC3-158P3D2, J82-158P3D2, 769-P-158P3D2 and CaKi-158P3D2 cell populations are generated by retroviral gene transfer as described in Hubert, R. S., et al., STEAP: a prostate-specific cell-surface antigen highly expressed in human prostate tumors. Proc Natl Acad Sci USA, 1999. 96(25): p. 14523-8. Anti-158P3D2 staining is detected by using an FITC-conjugated goat anti-mouse antibody (Southern Biotechnology Associates) followed by analysis on a Coulter Epics-XL f low cytometer.

Xenograft Mouse Models.

Subcutaneous (s.c.) tumors are generated by injection of 1×10⁶ AGS-K3, AGS-K6, A UMUC3-158P3D2, SCABER-158P3D2, 769-P-158P3D2 and CaKi-158P3D2 cells mixed at a 1:1 dilution with Matrigel (Collaborative Research) in the right flank of male SCID mice. To test antibody efficacy on tumor formation, i.p. antibody injections are started on the same day as tumor-cell injections. As a control, mice are injected with either purified mouse IgG (ICN) or PBS; or a purified monoclonal antibody that recognizes an irrelevant antigen not expressed in human cells. Tumor sizes are determined by vernier caliper measurements, and the tumor volume is calculated as length×width×height. Mice with s.c. tumors greater than 1.5 cm in diameter are sacrificed. PSA levels are determined by using a PSA ELISA kit (Anogen, Mississauga, Ontario). Circulating levels ofanti-158P3D2 mAbs are determined by a capture ELISA kit (Bethyl Laboratories, Montgomery, Tex.). (See, e.g., (Saffran, D., et al., PNAS 10:1073-1078.)

Orthotopic injections are performed under anesthesia by using ketamine/xylazine. For bladder orthotopic studies, an incision is made through the abdominal muscles to expose the bladder. Cells (5×10⁵) mixed with Matrigel are injected into the bladder in a 10-μl volume. For kidney orthopotic models, an incision is made through the abdominal muscles to expose the kidney. AGS-K3 or AGS-K6 cells mixed with Matrigel are injected under the kidney capsule. The mice are segregated into groups for the appropriate treatments, with anti-158P3D2 or control mAbs being injected i.p.

Anti-158P3D2 mAbs Inhibit Growth of 158P3D2-Expressing Xenograft-Cancer Tumors

The effect of anti-158P3D2 mAbs on tumor formation is tested by using bladder and kidney orthotopic models. As compared with the s.c. tumor model, the orthotopic model, which requires injection of tumor cells directly in the mouse bladder or kidney, respectively, results in a local tumor growth, development of metastasis in distal sites, deterioration of mouse health, and subsequent death (Saffran, D., et al., PNAS supra; Fu, X., et al., Int J Cancer, 1992. 52(6): p. 987-90; Kubota, T., J Cell Biochem, 1994. 56(1): p. 4-8). The features make the orthotopic model more representative of human disease progression and allowed us to follow the therapeutic effect of mAbs on clinically relevant end points.

Accordingly, tumor cells are injected into the mouse bladder or kidney, and 2 days later, the mice are segregated into two groups and treated with either: a) 200-500 μg, of anti-158P3D2 Ab, or b) PBS three times per week for two to five weeks.

A major advantage of the orthotopic cancer model is the ability to study the development of metastases. Formation of metastasis in mice bearing established orthotopic tumors is studies by IHC analysis on lung sections using an antibody against BTA, a bladder specific antigen (Hubert, R. S., et al., Proc Natl Acad Sci USA, 1999. 96(25): p. 14523-8) or anti-G250 antibody for kidney cancer models.

Mice bearing established orthotopic tumors are administered 1000 μg injections of either anti-158P3D2 mAb or PBS over a 4-week period. Mice in both groups are allowed to establish a high tumor burden, to ensure a high frequency of metastasis formation in mouse lungs. Mice then are killed and their bladder/kidney and lungs are analyzed for the presence of tumor cells by IHC analysis.

These studies demonstrate a broad anti-tumor efficacy of anti-158P3D2 antibodies on initiation and progression of bladder and kidney cancer in xenograft mouse models. Anti-158P3D2 antibodies inhibit tumor formation of both androgen-dependent and androgen-independent tumors as well as retarding the growth of already established tumors and prolong the survival of treated mice. Moreover, anti-158P3D2 mAbs demonstrate a dramatic inhibitory effect on the spread of local prostate tumor to distal sites, even in the presence of a large tumor burden. Thus, anti-158P3D2 mAbs are efficacious on major clinically relevant end points (tumor growth), prolongation of survival, and health.

Example 39 Therapeutic and Diagnostic Use of Anti-158P3D2 Antibodies in Humans

Anti-158P3D2 monoclonal antibodies are safely and effectively used for diagnostic, prophylactic, prognostic and/or therapeutic purposes in humans. Western blot and immunohistochemical analysis of cancer tissues and cancer xenografts with anti-158P3D2 mAb show strong extensive staining in carcinoma but significantly lower or undetectable levels in normal tissues. Detection of 158P3D2 in carcinoma and in metastatic disease demonstrates the usefulness of the mAb as a diagnostic and/or prognostic indicator. Anti-158P3D2 antibodies are therefore used in diagnostic applications such as immunohistochemistry of kidney biopsy specimens to detect cancer from suspect patients.

As determined by flow cytometry, anti-158P3D2 mAb specifically binds to carcinoma cells. Thus, anti-158P3D2 antibodies are used in diagnostic whole body imaging applications, such as radioimmunoscintigraphy and radioimmunotherapy, (see, e.g., Potamianos S., et. al. Anticancer Res 20(2A):925-948 (2000)) for the detection of localized and metastatic cancers that exhibit expression of 158P3D2. Shedding or release of an extracellular domain of 158P3D2 into the extracellular milieu, such as that seen for alkaline phosphodiesterase BIO (Meerson, N. R., Hepatology 27:563-568 (1998)), allows diagnostic detection of 158P3D2 by anti-158P3D2 antibodies in serum and/or urine samples from suspect patients.

Anti-158P3D2 antibodies that specifically bind 158P3D2 are used in therapeutic applications for the treatment of cancers that express 158P3D2. Anti-158P3D2 antibodies are used as an unconjugated modality and as conjugated form in which the antibodies are attached to one of various therapeutic or imaging modalities well known in the art, such as a prodrugs, enzymes or radioisotopes. In preclinical studies, unconjugated and conjugated anti-158P3D2 antibodies are tested for efficacy of tumor prevention and growth inhibition in the SCID mouse cancer xenograft models, e.g., kidney cancer models AGS-K3 and AGS-K6, (see, e.g., Example 38). Conjugated and unconjugaied anti-158P3D2 antibodies are used as a therapeutic modality in human clinical trials either alone or in combination with other treatments as described in following Examples.

Example 40 Human Clinical Trials for the Treatment and Diagnosis of Human Carcinomas Through use of Human Anti-158P3D2 Antibodies In Vivo

Antibodies are used in accordance with the present invention which recognize an epitope on 158P3D2, and are used in the treatment of certain tumors such as those listed in Table I. Based upon a number of factors, including 158P3D2 expression levels, tumors such as those listed in Table I are presently preferred indications. In connection with each of these indications, three clinical approaches are successfully pursued.

I.) Adjunctive therapy: In adjunctive therapy, patients are treated with anti-158P3D2 antibodies in combination with a chemotherapeutic or antineoplastic agent and/or radiation therapy. Primary cancer targets, such as those listed in Table I, are treated under standard protocols by the addition anti-158P3D2 antibodies to standard first and second line therapy. Protocol designs address effectiveness as assessed by reduction in tumor mass as well as the ability to reduce usual doses of standard chemotherapy. These dosage reductions allow additional and/or prolonged therapy by reducing dose-related toxicity of the chemotherapeutic agent. Anti-158P3D2 antibodies are utilized in several adjunctive clinical trials in combination with the chemotherapeutic or antineoplastic agents adriamycin (advanced prostrate carcinoma), cisplatin (advanced head and neck and lung carcinomas), taxol (breast cancer), and doxorubicin (preclinical).

II.) Monotherapy: In connection with the use of the anti-158P3D2 antibodies in monotherapy of tumors, the antibodies are administered to patients without a chemotherapeutic or antineoplastic agent. In one embodiment, monotherapy is conducted clinically in end stage cancer patients with extensive metastatic disease. Patients show some disease stabilization. Trials demonstrate an effect in refractory patients with cancerous tumors.

III.) Imaging Agent: Through binding a radionuclide (e.g., iodine or yttrium (I¹³¹, Y⁹⁰) to anti-158P3D2 antibodies, the radiolabeled antibodies are utilized as a diagnostic and/or imaging agent. In such a role, the labeled antibodies localize to both solid tumors, as well as, metastatic lesions of cells expressing 158P3D2. In connection with the use of the anti-158P3D2 antibodies as imaging agents, the antibodies are used as an adjunct to surgical treatment of solid tumors, as both a pre-surgical screen as well as a post-operative follow-up to determine what tumor remains and/or returns. In one embodiment, a (¹¹¹In)-158P3D2 antibody is used as an imaging agent in a Phase I human clinical trial in patients having a carcinoma that expresses 158P3D2 (by analogy see, e.g., Divgi et al. J. Natl. Cancer Inst 83:97-104 (1991)). Patients are followed with standard anterior and posterior gamma camera. The results indicate that primary lesions and metastatic lesions are identified

Dose and Route of Administration

As appreciated by those of ordinary skill in the art, dosing considerations can be determined through comparison with the analogous products that are in the clinic. Thus, anti-158P3D2 antibodies can be administered with doses in the range of 5 to 400 mg/m², with the lower doses used, e.g., in connection with safety studies. The affinity of anti-158P3D2 antibodies relative to the affinity of a known antibody for its target is one parameter used by those of skill in the art for determining analogous dose regimens. Further, anti-158P3D2 antibodies that are fully human antibodies, as compared to the chimeric antibody, have slower clearance; accordingly, dosing in patients with such fully human anti-158P3D2 antibodies can be lower, perhaps in the range of 50 to 300 mg/m², and still remain efficacious. Dosing in mg/m², as opposed to the conventional measurement of dose in mg/kg, is a measurement based on surface area and is a convenient dosing measurement that is designed to include patients of all sizes from infants to adults.

Three distinct delivery approaches are useful for delivery of anti-158P3D2 antibodies. Conventional intravenous delivery is one standard delivery technique for many tumors. However, in connection with tumors in the peritoneal cavity, such as tumors of the ovaries, biliary duct, other ducts, and the like, intraperitoneal administration may prove favorable for obtaining high dose of antibody at the tumor and to also minimize antibody clearance. In a similar manner, certain solid tumors possess vasculature that is appropriate for regional perfusion. Regional perfusion allows for a high dose of antibody at the site of a tumor and minimizes short term clearance of the antibody.

Clinical Development Plan (CDP)

Overview: The CDP follows and develops treatments of anti-158P3D2 antibodies in connection with adjunctive therapy, monotherapy, and as an imaging agent. Trials initially demonstrate safety and thereafter confirm efficacy in repeat doses. Trails are open label comparing standard chemotherapy with standard therapy plus anti-158P3D2 antibodies. As will be appreciated, one criteria that can be utilized in connection with enrollment of patients is 158P3D2 expression levels in their tumors as determined by biopsy.

As with any protein or antibody infusion-based therapeutic, safety concerns are related primarily to (i) cytokine release syndrome, i.e., hypotension, fever, shaking, chills; (ii) the development of an immunogenic response to the material (i.e., development of human antibodies by the patient to the antibody therapeutic, or HAHA response); and, (iii) toxicity to normal cells that express 158P3D2. Standard tests and follow-up are utilized to monitor each of these safety concerns. Anti-158P3D2 antibodies are found to be safe upon human administration.

Example 41 Human Clinical Trial Adjunctive Therapy with Human Anti-158P3D2 Antibody and Chemotherapeutic Agent

A phase I human clinical trial is initiated to assess the safety of six intravenous doses of a human anti-158P3D2 antibody in connection with the treatment of a solid tumor, e.g., a cancer of a tissue listed in Table I. In the study, the safety of single doses of anti-158P3D2 antibodies when utilized as an adjunctive therapy to an antineoplastic or chemotherapeutic agent, such as cisplatin, topotecan, doxorubicin, adriamycin, taxol, or the like, is assessed. The trial design includes delivery of six single doses of an anti-158P3D2 antibody with dosage of antibody escalating from approximately about 25 mg/m² to about 275 mg/m² over the course of the treatment in accordance with the following schedule:

Day 0 Day 7 Day 14 Day 21 Day 28 Day 35 mAb Dose 25 mg/m² 75 mg/m² 125 mg/m² 175 mg/m² 225 mg/m² 275 mg/m² Chemotherapy + + + + + + (standard dose)

Patients are closely followed for one-week following each administration of antibody and chemotherapy. In particular, patients are assessed for the safety concerns mentioned above: (i) cytokine release syndrome, i.e., hypotension, fever, shaking, chills; (ii) the development of an immunogenic response to the material (i.e., development of human antibodies by the patient to the human antibody therapeutic, or HAHA response); and, (iii) toxicity to normal cells that express 158P3D2. Standard tests and follow-up are utilized to monitor each of these safety concerns. Patients are also assessed for clinical outcome, and particularly reduction in tumor mass as evidenced by MRI or other imaging.

The anti-158P3D2 antibodies are demonstrated to be safe and efficacious, Phase II trials confirm the efficacy and refine optimum dosing.

Example 42 Human Clinical Trial: Monotherapy with Human Anti-158P3D2 Antibody

Anti-158P3D2 Antibodies are Safe in Connection with the Above-Discussed Adjunctive Trial, a Phase II Human Clinical Trial Confirms the Efficacy and Optimum Dosing for Monotherapy. Such Trial is Accomplished, and Entails the Same Safety and Outcome Analyses, to the Above-Described Adjunctive Trial with the Exception being that Patients do not Receive Chemotherapy Concurrently with the Receipt of Doses of Anti-158P3D2 Antibodies.

Example 43 Human Clinical Trial Diagnostic Imaging with Anti-158P3D2 Antibody

Once again, as the adjunctive therapy discussed above is safe within the safety criteria discussed above, a human clinical trial is conducted concerning the use of anti-158P3D2 antibodies as a diagnostic imaging agent. The protocol is designed in a substantially similar manner to those described in the art, such as in Divgi et al. J. Natl. Cancer Inst. 83:97-104 (1991). The antibodies are found to be both safe and efficacious when used as a diagnostic modality.

Example 44 Homology Comparison of 158P3D2 to Known Sequences

The 158P3D2 gene is identical to a previously cloned and sequenced gene, namely a novel protein similar to otoferlin and dysferlin, isoform 1 (gi 7671662), showing 100% identity to that protein (FIG. 4B). The 158P3D2 protein shows 65% homology and 45% identity to human otoferlin long isoform (gi 10119916), and 45% identity and 45% homology to the mouse otoferlin (gi 13994207) (FIGS. 4C and 4D, respectively). The 158P3D2 protein consists of 328 amino acids, with calculated molecular weight of 38.4 kDa, and pI of 8.64. 158P3D2 is a cell surface protein, with possible localization to the endoplasmic reticulum fraction. The 158P3D2 protein contains a single transmembrane domain at aa 145. Motif analysis revealed the presence of several known motifs, including a C2 domains located at the amino acids 122-144 of the 158P3D2 protein, an aminoacyl-transfer RNA synthetases class II motif at aa 91-115. Pfam analysis suggests that 158P3D2 has a slight likelihood of belonging to the chemokine receptor family (Table XXII).

C2 domains are Ca2+-binding motifs present in a variety of proteins including phospholipases, protein kinases C and synaptotamins (Murakami M, et al Biochim Biophys Acta. 2000, 1488:159; Marqueze B et al, Biochimie. 2000, 82:409). They are about 116 amino-acid residues long, and function in calcium-dependent phospholipid binding (Stahelin R V, Cho W. Biochem J. 2001, 359:679). Since some C2-related domains are found in proteins that do not bind calcium, C2 domains have been assigned an additional function, namely inter-molecular association, such as binding to inositol-1,3,4,5-tetraphosphate (Mehrotra B et al, Biochemistry. 2000, 39:9679). C2 domains are also instrumental in targeting proteins to specific subcellular locations. In particular, recent studies have shown that the C2 domain of PLA mediates the translocation of PLA from the cytosol to the golgi in response to calcium (Evans J H et al, J Biol Chem. 2001, 276:30150). In addition to affecting localization and protein association, C2 domain proteins have been reported to regulate critical cellular functions, including proliferation, a key component of tumoriogenesis (Koehler J A, Moran M F. Cell Growth Differ. 2001, 12:551).

Aminoacyl-tRNA synthetases are enzymes that activate amino acids and transfer them to specific tRNA molecules as the first step in protein biosynthesis (Fabrega C et al, Nature. 2001, 411:110). In eukaryotes two aminoacyl-tRNA synthetases exist for each of the 20 essential amino acid: a cytosolic form and a mitochondrial form. The class II synthetases are specific for alanine, asparagine, aspartic acid, glycine, histidine, lysine, phenylalanine, proline, serine, and threonine. Since aminoacyl transfer RNA synthetases regulate protein synthesis, it is clear that they also regulate cell proliferation and maintain the accuracy of protein synthesis (Jakubowski H, Goldman E. Microbiol Rev. 1992, 56:412). This characteristic of aminoacyl transfer RNA synthetases was used to develop reagents with anti-tumor effects in vitro (Laske R et al, Arch Pharm. 1991, 324:153). The relevance of aminoacyl transfer RNA synthetases to cell survival and growth was demonstrated in cells expressing mutant lysyl-tRNA synthetase. Mutation in lysyl-tRNA synthetases resulted in apoptosis of BHK21 cells (Fukushima et al, Genes Cells. 1996, 1:1087).

Based on the information above, 158P3D2 plays an important role in several biological processes, including protein synthesis, cell growth, metabolism, and survival.

Several isoforms of 158P3D2 have been identified (FIG. 11). While both variants var2a and var5a do not contain a transmembrane domain, var2a still maintains the C2 domain important for protein interaction, localization and calcium binding. Variant var2b still maintains the transmembrane domain, but fails to exhibit a well-identified C2 domains. In addition, two variants, var3 and var4 contain a point mutations at amino acid 103 and 102, respectively, relative to the 158P3D2 var1 protein. These single amino acid changes do not significantly alter the predicted localization or motifs associated with 158P3D2 var1.

Accordingly, when any of the 158P3D2 variants function as regulators of protein synthesis, cell growth, metabolism, and survival, 158P3D2 is used for therapeutic, diagnostic, prognostic and/or preventative purposes.

Example 45 Identification and Confirmation of Potential Signal Transduction Pathways

Many mammalian proteins have been reported to interact with signaling molecules and to participate in regulating signaling pathways (J Neurochem. 2001; 76:217-223). In particular, C2-domain containing proteins have been reported to associate with signaling molecules and regulate signaling pathways including mitogenic cascades (Chow A et al, FEBS Lett. 2000; 469:88; Walker E H et al, Nature. 1999, 402:313). Using immunoprecipitation and Western blotting techniques, proteins are identified that associate with 158P3D2 and mediate signaling events. Several pathways known to play a role in cancer biology can be regulated by 158P3D2, including phospholipid pathways such as PI3K, AKT, etc, adhesion and migration pathways, including FAK, Rho, Rac-1, etc, as well as mitogenic/survival cascades such as ERK, p38, etc (Cell Growth Differ. 2000, 11:279; J Biol Chem. 1999, 274:801; Oncogene. 2000, 19:3003, J. Cell Biol. 1997, 138:913.).

To confirm that 158P3D2 directly or indirectly activates known signal transduction pathways in cells, luciferase (luc) based transcriptional reporter assays are carried out in cells expressing individual genes. These transcriptional reporters contain consensus-binding sites for known transcription factors that lie downstream of well-characterized signal transduction pathways. The reporters and examples of these associated transcription factors, signal transduction pathways, and activation stimuli are listed below.

-   -   1. NFkB-luc, NFkB/Rel; Ik-kinase/SAPK; growth/apoptosis/stress     -   2. SRE-luc, SRF/TCF/ELK1; MAPK/SAPK; growth/differentiation     -   3. AP-1-luc, FOS/JUN; MAPK/SAPK/PKC; growth/apoptosis/stress     -   4. ARE-luc, androgen receptor; steroids/MAPK;         growth/differentiation/apoptosis     -   5. p53-luc, p53; SAPK; growth/differentiation/apoptosis     -   6. CRE-luc, CREB/ATF2; PKA/p38; growth/apoptosis/stress

Gene-mediated effects can be assayed in cells showing mRNA expression. Luciferase reporter plasmids can be introduced by lipid-mediated transfection (TFX-50, Promega). Luciferase activity, an indicator of relative transcriptional activity, is measured by incubation of cell extracts with luciferin substrate and luminescence of the reaction is monitored in a luminometer.

Signaling pathways activated by 158P3D2 are mapped and used for the identification and validation of therapeutic targets. When 158P3D2 is involved in cell signaling, it is used as target for diagnostic, prognostic, preventative and/or therapeutic purposes.

Example 46 Involvement in Tumor Progression

Based on the reported effect of C2 domains and tRNA synthetases on cell growth, survival, protein regulation and signaling, the 158P3D2 gene can contribute to the growth of cancer cells. The role of 158P3D2 in tumor growth is confirmed in a variety of primary and transfected cell lines including, bladder and kidney cell lines, as well as NIH 3T3 cells engineered to stably express 158P3D2. Parental cells lacking 158P3D2 and cells expressing 158P3D2 are evaluated for cell growth using a well-documented proliferation assay (Fraser S P, Grimes J A, Djamgoz M B. Prostate. 2000; 44:61, Johnson D E, Ochieng J, Evans S L. Anticancer Drugs. 1996, 7:288).

To confirm the role of 158P3D2 in the transformation process, its effect in colony forming assays is investigated. Parental NIH-3T3 cells lacking 158P3D2 are compared to NIH-3T3 cells expressing 158P3D2, using a soft agar assay under stringent and more permissive conditions (Song Z. et al. Cancer Res. 2000; 60:6730).

To confirm the role of 158P3D2 in invasion and metastasis of cancer cells, a well-established assay is used, e.g., a Transwell Insert System assay (Becton Dickinson) (Cancer Res. 1999; 59:6010). Control cells, including bladder and kidney cell lines lacking 158P3D2 are compared to cells expressing 158P3D2. Cells are loaded with the fluorescent dye, calcein, and plated in the top well of the Transwell insert coated with a basement membrane analog. Invasion is determined by fluorescence of cells in the lower chamber relative to the fluorescence of the entire cell population. 158P3D2 can also play a role in cell cycle and apoptosis. Parental cells and cells expressing

158P3D2 are compared for differences in cell cycle regulation using a well-established BrdU assay (Abdel-Malek Z A. J Cell Physiol. 1988, 136:247). In short, cells are grown under both optimal (full serum) and limiting (low serum) conditions are labeled with BrdU and stained with anti-BrdU Ab and propidium iodide. Cells are analyzed for entry into the G1, S, and G2M phases of the cell cycle. Alternatively, the effect of stress on apoptosis is evaluated in control parental cells and cells expressing 158P3D2, including normal and tumor prostate, colon and lung cells. Engineered and parental cells are treated with various chemotherapeutic agents, such as etoposide, flutamide, etc, and protein synthesis inhibitors, such as cycloheximide. Cells are stained with annexin V-FITC and cell death is measured by FACS analysis. The modulation of cell death by 158P3D2 can play a critical role in regulating tumor progression and tumor load.

When 158P3D2 plays a role in cell growth, transformation, invasion or apoptosis, it is used as a target for diagnostic, prognostic, preventative and/or therapeutic purposes.

Example 47 Involvement in Angiogenesis

Angiogenesis or new capillary blood vessel formation is necessary for tumor growth (Hanahan D, Folkman J. Cell. 1996, 86:353; Folkman J. Endocrinology. 1998 139:441). Based on the effect of 158P3D2 on cellular functions and protein expression, 158P3D2 plays a role in angiogenesis. In addition, recent studies have associated human tyrosyl- and tryptophanyl-tRNA synthetases to angiogenesis (Otani A et al, Proc Natl Acad Sci USA. 2002, 99:178). Several assays have been developed to measure angiogenesis in vitro and in vivo, such as the tissue culture assays endothelial cell tube formation and endothelial cell proliferation. Using these assays as well as in vitro neo-vascularization, the role of 158P3D2 in angiogenesis, enhancement or inhibition, is confirmed.

For example, endothelial cells engineered to express 158P3D2 are evaluated using tube formation and proliferation assays. The effect of 158P3D2 is also confirmed in animal models in vivo. For example, cells either expressing or lacking 158P3D2 are implanted subcutaneously in immunocompromised mice. Endothelial cell migration and angiogenesis are evaluated 5-15 days later using immunohistochemistry techniques. 158P3D2 affects angiogenesis, and it is used as a target for diagnostic, prognostic, preventative and/or therapeutic purposes

Example 48 Regulation of Protein Synthesis

The presence of a tRNA synthetase motif indicates that 158P3D2 regulates protein synthesis. Regulation of protein synthesis is confirmed, e.g., by studying gene expression in cells expressing or lacking 158P3D2. For this purpose, cells are labeled with ³H-Leucine and evaluated for the incorporation of the isotope (Tsurusaki Y, Yamaguchi M. Int J Mol Med. 2000, 6:295). For examples cells lacking or expressing 158P3D2 are incubated with ³H-Leucine for 6 hours in the presence of absence of stimuli such as growth factors, serum, phorbol esters. Cells are lysed and evaluated for ³H-Leucine incorporation using a beta-counter (cpm).

Thus, 158P3D2 regulates protein synthesis, it is used as a target for diagnostic, prognostic, preventative and/or therapeutic purposes.

Example 49 Protein-Protein Association

C2 domain-containing proteins have been shown to mediate protein-protein association (Murakami M, et al Biochim Biophys Acta. 2000, 1488:159; Chow A et al, FEBS Lett. 2000; 469:88). Using immunoprecipitation techniques as well as two yeast hybrid systems, proteins are identified that associate with 158P3D2. Immunoprecipitates from cells expressing 158P3D2 and cells lacking 158P3D2 are compared for specific protein-protein associations.

Studies are performed to confirm the extent of association of 158P3D2 with effector molecules, such as signaling intermediates, nuclear proteins, transcription factors, kinases, phosophates, etc. Studies comparing 158P3D2 positive and 158P3D2 negative cells as well as studies comparing unstimulated/resting cells and cells treated with epithelial cell activators, such as cytokines, growth factors, androgen and anti-integrin Ab reveal unique interactions.

In addition, protein-protein interactions are confirmed using two yeast hybrid methodology (Curr Opin Chem Biol. 1999, 3:64). A vector carrying a library of proteins fused to the activation domain of a transcription factor is introduced into yeast expressing a 158P3D2-DNA-binding domain fusion protein and a reporter construct. Protein-protein interaction is detected by calorimetric reporter activity. Specific association with effector molecules and transcription factors directs one of skill to the mode of action of 158P3D2, and thus identifies therapeutic, prognostic, preventative and/or diagnostic targets for cancer. This and similar assays are also used to identify and screen for small molecules that interact with 158P3D2.

Thus it is found that 158P3D2 associates with proteins and small molecules. Accordingly, 158P3D2 and these proteins and small molecules are used for diagnostic, prognostic, preventative and/or therapeutic purposes.

Throughout this application, various website data content, publications, patent applications and patents are referenced. (Websites are referenced by their Uniform Resource Locator, or URL, addresses on the World Wide Web.) The disclosures of each of these references are hereby incorporated by reference herein in their entireties.

The present invention is not to be limited in scope by the embodiments disclosed herein, which are intended as single illustrations of individual aspects of the invention, and any that are functionally equivalent are within the scope of the invention. Various modifications to the models and methods of the invention, in addition to those described herein, will become apparent to those skilled in the art from the foregoing description and teachings, and are similarly intended to fall within the scope of the invention. Such modifications or other embodiments can be practiced without departing from the true scope and spirit of the invention.

Tables

TABLE I Tissues that Express 158P3D2 When Malignant Prostate Bladder Kidney Colon Ovary Lung Breast Pancreas

TABLE II Amino Acid Abbreviations SINGLE LETTER THREE LETTER FULL NAME F Phe phenylalanine L Leu leucine S Ser serine Y Tyr tyrosine C Cys cysteine W Trp tryptophan P Pro proline H His histidine Q Gln glutamine R Arg arginine I Ile isoleucine M Met methionine T Thr threonine N Asn asparagine K Lys lysine V Val valine A Ala alanine D Asp aspartic acid E Glu glutamic acid G Gly glycine

TABLE III Amino Acid Substitution Matrix Adapted from the GCG Software 9.0 BLOSUM62 amino acid substitution matrix (block substitution matrix). The higher the value, the more likely a substitution is found in related, natural proteins. A C D E F G H I K L M N P Q R S T V W Y . 4 0 −2 −1 −2 0 −2 −1 −1 −1 −1 −2 −1 −1 −1 1 0 0 −3 −2 A 9 −3 −4 −2 −3 −3 −1 −3 −1 −1 −3 −3 −3 −3 −1 −1 −1 −2 −2 C 6 2 −3 −1 −1 −3 −1 −4 −3 1 −1 0 −2 0 −1 −3 −4 −3 D 5 −3 −2 0 −3 1 −3 −2 0 −1 2 0 0 −1 −2 −3 −2 E 6 −3 −1 0 −3 0 0 −3 −4 −3 −3 −2 −2 −1 1 3 F 6 −2 −4 −2 −4 −3 0 −2 −2 −2 0 −2 −3 −2 −3 G 8 −3 −1 −3 −2 1 −2 0 0 −1 −2 −3 −2 2 H 4 −3 2 1 −3 −3 −3 −3 −2 −1 3 −3 −1 I 5 −2 −1 0 −1 1 2 0 −1 −2 −3 −2 K 4 2 −3 −3 −2 −2 −2 −1 1 −2 −1 L 5 −2 −2 0 −1 −1 −1 1 −1 −1 M 6 −2 0 0 1 0 −3 −4 −2 N 7 −1 −2 −1 −1 −2 −4 −3 P 5 1 0 −1 −2 −2 −1 Q 5 −1 −1 −3 −3 −2 R 4 1 −2 −3 −2 S 5 0 −2 −2 T 4 −3 −1 V 11 2 W 7 Y

TABLE IV HLA Class I/II Motifs/Supermotifs TABLE IV (A): HLA Class I Supermotifs/Motifs POSITION POSITION POSITION C Terminus (Primary 2 (Primary Anchor) 3 (Primary Anchor) Anchor) SUPERMOTIFS A1 TI LVMS FWY A2 LIVM ATQ IV MATL A3 VSMA TLI RK A24 YF WIVLMT FI YWLM B7 P VILF MWYA B27 RHK FYL WMIVA B44 E D FWYLIMVA B58 ATS FWY LIVMA B62 QL IVMP FWYMIVLA MOTIFS A1 TSM Y A1 DE AS Y A2.1 LM VQIAT V LIMAT A3 LMVISATF CGD KYR HFA A11 VTMLISAGN CDF K RYH A24 YFW M FLIW A*3101 MVT ALIS R K A*3301 MVALF IST RK A*6801 AVT MSLI RK B*0702 P LMF WYAIV B*3501 P LMFWY IVA B51 P LIVF WYAM B*5301 P IMFWY ALV B*5401 P ATIV LMFWY TABLE IV (B): HLA Class II Supermotif 1 6 9 W, F, Y, V, .I, L A, V, I, L, P, C, S, T A, V, I, L, C, S, T, M, Y TABLE IV (C): HLA Class II Motifs MOTIFS 1° anchor 1 2 3 4 5 1° anchor 6 7 8 9 DR4 preferred FMYLIVW M T I VSTCPALIM MH MH deleterious W R WDE DR1 preferred MFLIVWY PAMQ VMATSPLIC M AVM deleterious C CH FD CWD GDE D DR7 preferred MFLIVWY M W A IVMSACTPL M IV deleterious C G GRD N G DR3 MOTIFS 1° anchor 1 2 3 1° anchor 4 5 1° anchor 6 motif a LIVMFY D preferred motif b LIVMFAY DNQEST KRH preferred DR MFLIVWY VMSTACPLI Supermotif TABLE IV (D): HLA Class I Supermotifs SUPER- POSITION: MOTIFS 1 2 3 4 5 6 7 8 C-terminus A1 1° Anchor 1° Anchor TILVMS FWY A2 1° Anchor 1° Anchor LIVMATQ LIVMAT A3 preferred 1° Anchor YFW YFW YFW P 1° Anchor VSMATLI (4/5) (3/5) (4/5) (4/5) RK deleterious DE (3/5); DE P (5/5) (4/5) A24 1° Anchor 1° Anchor YFWIVLMT FIYWLM B7 preferred FWY (5/5) 1° Anchor FWY FWY 1° Anchor LIVM (3/5) P (4/5) (3/5) VILFMWYA deleterious DE (3/5); DE G QN DE P (5/5); (3/5) (4/5) (4/5) (4/5) G (4/5); A (3/5); QN (3/5) B27 1° Anchor 1° Anchor RHK FYLWMIVA B44 1° Anchor 1° Anchor ED FWYLIMVA B58 1° Anchor 1° Anchor ATS FWYLIVMA B62 1° Anchor 1° Anchor QLIVMP FWYMIVLA TABLE IV (E): HLA Class I Motifs POSITION: 1 2 3 4 5 6 A1 preferred GFYW 1° Anchor DEA YFW P 9-mer STM deleterious DE RHKLIVMP A G A A1 preferred GRHK ASTCLIVM 1° Anchor GSTC ASTC 9-mer DEAS deleterious A RHKDEPY DE PQN RHK FW A1 preferred YFW 1° Anchor DEAQN A YFWQN 10-mer STM deleterious GP RHKGLIVM DE RHK QNA A1 preferred YFW STCLIVM 1° Anchor A YFW 10-mer DEAS deleterious RHK RHKDEPY P G FW A2.1 preferred YFW 1° Anchor YFW STC YFW 9-mer LMIVQAT deleterious DEP DERKH RKH A2.1 preferred AYFW 1° Anchor LVIM G G 10-mer LMIVQAT deleterious DEP DE RKHA P A3 preferred RHK 1° Anchor YFW PRHKYFW A YFW LMVISA TFCGD deleterious DEP DE A11 preferred A 1° Anchor YFW YFW A YFW VTLMIS AGNCDF deleterious DEP A24 preferred YFWRHK 1° Anchor STC 9-mer YFWM deleterious DEG DE G QNP DERHK A24 preferred 1° Anchor P YFWP 10-mer YFWM deleterious GDE QN RHK DE A3101 preferred RHK 1° Anchor YFW P YFW MVTALIS deleterious DEP DE ADE DE A3301 preferred 1° Anchor YFW MVALFIST deleterious GP DE A6801 preferred YFWSTC 1° Anchor YFWLIVM AVTMSLI deleterious GP DEG RHK B0702 preferred RHKFWY 1° Anchor RHK RHK RHK P deleterious DEQNP DEP DE DE GDE B3501 preferred FWYLIVM 1° Anchor FWY P deleterious AGP G G B51 preferred LIVMFWY 1° Anchor FWY STC FWY P deleterious AGPDER DE G HKSTC B5301 preferred LIVMFWY 1° Anchor FWY STC FWY P deleterious AGPQN G B5401 preferred FWY 1° Anchor FWYL LIVM P IVM deleterious GPQNDE GDES RHKDE DE TC POSITION: 9 or 7 8 C-terminus C-terminus A1 preferred DEQN YFW 1° Anchor 9-mer Y deleterious A1 preferred LIVM DE 1° Anchor 9-mer Y deleterious PG GP A1 preferred PASTC GDE P 1° Anchor 10-mer Y deleterious RHKYFW RHK A A1 preferred PG G YFW 1° Anchor 10-mer Y deleterious PRHK QN A2.1 preferred A P 1° Anchor 9-mer VLIMAT deleterious DERKH A2.1 preferred FYWL 1° Anchor 10-mer VIM VLIMAT deleterious RKH DERKH RKH A3 preferred P 1° Anchor KYRHFA deleterious A11 preferred YFW P 1° Anchor KRYH deleterious A G A24 preferred YFW YFW 1° Anchor 9-mer FLIW deleterious G AQN A24 preferred P 1° Anchor 10-mer FLIW deleterious A QN DEA A3101 preferred YFW AP 1° Anchor RK deleterious DE DE A3301 preferred AYFW 1° Anchor RK deleterious A6801 preferred YFW P 1° Anchor RK deleterious A B0702 preferred RHK PA 1° Anchor LMFWYAIV deleterious QN DE B3501 preferred FWY 1° Anchor LMFWYIVA deleterious B51 preferred G FWY 1° Anchor LIVFWYAM deleterious DEQN GDE B5301 preferred LIVMFWY FWY 1° Anchor IMFWYALV deleterious RHKQN DE B5401 preferred ALIVM FWYAP 1° Anchor ATIVLMF WY deleterious QNDGE DE Bolded residues are preferred, italicized residues are less preferred: A peptide is considered motif-bearing if it has primary anchors at each primary anchor position for a motif or supermotif as specified in the above table. Italicized residues indicate less preferred or “tolerated” residues. The information in this Table is specific for 9-mers unless otherwise specified.

TABLE V 158P3D2 A1, 9mers (variants 1, 2a, 3, 4 and 5a) 158P3D2 v.1 A1-9-mers SEQ. Pos 123456789 Score ID NO. 222 FTDMGGNVY 62.500 17  47 TGEMSSDIY 11.250 18 219 DLEFTDMGG 4.500 19 110 ALEEAEFRQ 4.500 20 237 EAEFELLTV 4.500 21 247 EAEKRPVGK 3.600 22 198 AQEAQAGKK 2.700 23  78 TGEGNFNWR 2.250 24 259 QPEPLEKPS 2.250 25 113 EAEFRQPAV 1.800 26 140 SLELQLPDM 1.800 27 281 KTFVFFIWR 1.250 28 303 LTVFLLLVF 1.250 29 145 LPDMVRGAR 1.250 30 312 YTIPGQISQ 1.250 31  69 ETDVHFNSL 1.250 32  34 NTEDVVLDD 1.125 33 320 QVIFRPLHK 1.000 34 166 GAGPRCNLF 1.000 35 304 TVFLLLVFY 1.000 36  39 VLDDENPLT 1.000 37 188 LKEAEDVER 0.900 38 235 KVEAEFELL 0.900 39 190 EAEDVEREA 0.900 40  62 GLEHDKQET 0.900 41  51 SSDIYVKSW 0.750 42   2 WIDIFPQDV 0.500 43 257 RKQPEPLEK 0.500 44 142 ELQLPDMVR 0.500 45 283 FVFFIWRRY 0.500 46 121 VLVLQVWDY 0.500 47 156 ELCSVQLAR 0.500 48 154 GPELCSVQL 0.450 49  97 EREVSVWRR 0.450 50 242 LLTVEEAEK 0.400 51 197 EAQEAQAGK 0.400 52 243 LTVEEAEKR 0.250 53  90 RFDYLPTER 0.250 54  49 EMSSDIYVK 0.200 55   4 DIFPQDVPA 0.200 56  11 PAPPPVDIK 0.200 57 123 VLQVWDYDR 0.200 58  53 DIYVKSWVK 0.200 59 262 PLEKPSRPK 0.180 60  75 NSLTGEGNF 0.150 61  67 KQETDVHFN 0.135 62 126 VWDYDRISA 0.125 63 293 RTLVLLLLV 0.125 64  81 GNFNWRFVF 0.125 65 277 VNPLKTFVF 0.125 66  77 LTGEGNFNW 0.125 67 214 KGRPEDLEF 0.125 68 270 KTSFNWFVN 0.125 69  85 WRFVFRFDY 0.125 70  40 LDDENPLTG 0.125 71 216 RPEDLEFTD 0.113 72 298 LLLVLLTVF 0.100 73 200 EAQAGKKKR 0.100 74 170 RCNLFRCRR 0.100 75 109 FALEEAEFR 0.100 76 276 FVNPLKTFV 0.100 77 244 TVEEAEKRP 0.090 78  25 SYELRVVIW 0.090 79 193 DVEREAQEA 0.090 80 195 EREAQEAQA 0.090 81 132 ISANDFLGS 0.075 82 316 GQISQVIFR 0.075 83 105 RSGPFALEE 0.075 84  10 VPAPPPVDI 0.050 85  71 DVHFNSLTG 0.050 86 300 LVLLTVFLL 0.050 87 137 FLGSLELQL 0.050 88 232 LTGKVEAEF 0.050 89 294 TLVLLLLVL 0.050 90 301 VLLTVFLLL 0.050 91 302 LLTVFLLLV 0.050 92 227 GNVYILTGK 0.050 93 297 LLLLVLLTV 0.050 94 296 VLLLLVLLT 0.050 95 131 RISANDFLG 0.050 96 308 LLVFYTIPG 0.050 97 245 VEEAEKRPV 0.045 98 143 LQLPDMVRG 0.030 99  24 ISYELRVVI 0.030 100 201 AQAGKKKRK 0.030 101  50 MSSDIYVKS 0.030 102 116 FRQPAVLVL 0.025 103  46 LTGEMSSDI 0.025 104 191 AEDVEREAQ 0.025 105  95 PTEREVSVW 0.022 106  59 WVKGLEHDK 0.020 107 179 LRGWWPVVK 0.020 108 306 FLLLVFYTI 0.020 109 157 LCSVQLARN 0.020 110 230 YILTGKVEA 0.020 111 309 LVFYTIPGQ 0.020 112 299 LLVLLTVFL 0.020 113  17 DIKPRQPIS 0.020 114 295 LVLLLLVLL 0.020 115 158 CSVQLARNG 0.015 116 158P3D2 v.2a A1-9mers SEQ. Pos 123456789 Score ID NO. 180 ETELTVAVF 45.000 117 203 HIDLENRFY 25.000 118 101 FSEPQISRG 13.500 119 138 KADPYVVVS 10.000 120  46 SLEEEFNHF 9.000 121  93 YPESEAVLF 4.500 122   6 DSDGVNLIS 3.750 123 205 DLENRFYSH 1.800 124 167 FGEILELSI 1.125 125  24 EAEVKGTVS 0.900 126 194 GSDDLIGET 0.750 127  95 ESEAVLFSE 0.675 128  57 WLNVFPLYR 0.500 129  35 KAVATLKIY 0.500 130  53 HFEDWLNVF 0.450 131 109 GIPQNRPIK 0.400 132 153 DTKERYIPK 0.250 133 201 ETHIDLENR 0.250 134   1 MDDPGDSDG 0.250 135  73 GGEEEGSGH 0.225 136  22 QGEAEVKGT 0.225 137  37 VATLKIYNR 0.200 138  30 TVSPKKAVA 0.200 139 130 LAPADPNGK 0.200 140 129 NLAPADPNG 0.200 141  19 IQDQGEAEV 0.150 142  78 GSGHLVGKF 0.150 143 175 ISLPAETEL 0.150 144 216 ANCGLASQY 0.125 145 134 DPNGKADPY 0.125 146  77 EGSGHLVGK 0.100 147  59 NVFPLYRGQ 0.100 148 162 QLNPIFGEI 0.100 149 143 VVVSAGRER 0.100 150  91 LIYPESEAV 0.100 151 178 PAETELTVA 0.090 152 170 ILELSISLP 0.090 153 187 VFEHDLVGS 0.090 154  45 RSLEEEFNH 0.075 155 151 RQDTKERYI 0.075 156   9 GVNLISMVG 0.050 157  56 DWLNVFPLY 0.050 158  36 AVATLKIYN 0.050 159 182 ELTVAVFEH 0.050 160 132 PADPNGKAD 0.050 161 198 LIGETHIDL 0.050 162 169 EILELSISL 0.050 163 192 LVGSDDLIG 0.050 164 186 AVFEHDLVG 0.050 165  79 SGHLVGKFK 0.050 166  74 GEEEGSGHL 0.045 167  75 EEEGSGHLV 0.045 168 223 QYEVWVQQG 0.045 169 118 LLVRVYVVK 0.040 170  88 GSFLIYPES 0.030 171 173 LSISLPAET 0.030 172 195 SDDLIGETH 0.025 173 113 NRPIKLLVR 0.025 174 150 ERQDTKERY 0.025 175 108 RGIPQNRPI 0.025 176  29 GTVSPKKAV 0.025 177 100 LFSEPQISR 0.025 178   4 PGDSDGVNL 0.025 179  48 EEEFNHFED 0.022 180  16 VGEIQDQGE 0.022 181 199 IGETHIDLE 0.022 182  98 AVLFSEPQI 0.020 183 121 RVYVVKATN 0.020 184 220 LASQYEVWV 0.020 185  26 EVKGTVSPK 0.020 186 117 KLLVRVYVV 0.020 187  27 VKGTVSPKK 0.020 188 215 RANCGLASQ 0.020 189 106 ISRGIPQNR 0.015 190 221 ASQYEVWVQ 0.015 191 211 YSHHRANCG 0.015 192 228 VQQGPQEPF 0.015 193  85 KFKGSFLIY 0.013 194 112 QNRPIKLLV 0.013 195 177 LPAETELTV 0.013 196 110 IPQNRPIKL 0.013 197  11 NLISMVGEI 0.010 198 144 VVSAGRERQ 0.010 199  90 FLIYPESEA 0.010 200  12 LISMVGEIQ 0.010 201  99 VLFSEPQIS 0.010 202  15 MVGEIQDQG 0.010 203  81 HLVGKFKGS 0.010 204  82 LVGKFKGSF 0.010 205 191 DLVGSDDLI 0.010 206 184 TVAVFEHDL 0.010 207  20 QDQGEAEVK 0.010 208 185 VAVFEHDLV 0.010 209 176 SLPAETELT 0.010 210 219 GLASQYEVW 0.010 211  97 EAVLFSEPQ 0.010 212 154 TKERYIPKQ 0.009 213  69 GQDGGGEEE 0.007 214  13 ISMVGEIQD 0.007 215 115 PIKLLVRVY 0.005 216 158P3D2 v.3 A1-9mers SEQ. Pos 123456789 Score ID NO.   3 EREVSVRRR 0.450 217   1 PTEREVSVR 0.225 218   5 EVSVRRRSG 0.010 219   7 SVRRRSGPF 0.001 220   2 TEREVSVRR 0.001 221   4 REVSVRRRS 0.001 222   9 RRRSGPFAL 0.000 223   6 VSVRRRSGP 0.000 224   8 VRRRSGPFA 0.000 225 158P3D2 v.4 A1-9mers SEQ. Pos 123456789 Score ID NO.   4 EREVSIWRR 0.450 226   2 PTEREVSIW 0.022 227   6 EVSIWRRSG 0.010 228   1 LPTEREVSI 0.005 229   3 TEREVSIWR 0.003 230   7 VSIWRRSGP 0.002 231   8 SIWRRSGPF 0.001 232   5 REVSIWRRS 0.001 233   9 IWRRSGPFA 0.000 234 158P3D2 v.5a A1-9mers SEQ. Pos 123456789 Score ID NO.  16 SLDPWSCSY 250.000 235  28 CVGPGAPSS 0.200 236   8 YTASLPMTS 0.125 237  32 GAPSSALCS 0.050 238  43 AMGPGRGAI 0.050 239  14 MTSLDPWSC 0.025 240  27 WCVGPGAPS 0.020 241  36 SALCSWPAM 0.020 242  49 GAICFAAAA 0.020 243  37 ALCSWPAMG 0.020 244   2 VLQVWDYTA 0.020 245  39 CSWPAMGPG 0.015 246  15 TSLDPWSCS 0.015 247  22 CSYQTWCVG 0.015 248  20 WSCSYQTWC 0.015 249  10 ASLPMTSLD 0.015 250  35 SSALCSWPA 0.015 251  45 GPGRGAICF 0.013 252  21 SCSYQTWCV 0.010 253   1 LVLQVWDYT 0.010 254  40 SWPAMGPGR 0.010 255   9 TASLPMTSL 0.010 256  11 SLPMTSLDP 0.005 257  31 PGAPSSALC 0.005 258  38 LCSWPAMGP 0.005 259  48 RGAICFAAA 0.005 260  44 MGPGRGAIC 0.005 261  25 QTWCVGPGA 0.005 262   6 WDYTASLPM 0.003 263  41 WPAMGPGRG 0.003 264  29 VGPGAPSSA 0.003 265   5 VWDYTASLP 0.003 266  30 GPGAPSSAL 0.003 267  33 APSSALCSW 0.003 268  12 LPMTSLDPW 0.003 269  47 GRGAICFAA 0.003 270   4 QVWDYTASL 0.002 271  24 YQTWCVGPG 0.002 272   3 LQVWDYTAS 0.002 273   7 DYTASLPMT 0.001 274  13 PMTSLDPWS 0.001 275  42 PAMGPGRGA 0.001 276  17 LDPWSCSYQ 0.001 277  18 DPWSCSYQT 0.001 278  34 PSSALCSWP 0.000 279  23 SYQTWCVGP 0.000 280  26 TWCVGPGAP 0.000 281  19 PWSCSYQTW 0.000 282  46 PGRGAICFA 0.000 283

TABLE VI 158P3D2 A1, 10mers (variants 1, 2a, 3, 4 and 5a) 158P3D2 v.1 A1-10mers SEQ. ID Pos 1234567890 Score NO. 259 QPEPLEKPSR 45.000 284 276 FVNPLKTFVF 5.000 285 166 GAGPRCNLFR 5.000 286 235 KVEAEFELLT 4.500 287 198 AQEAQAGKKK 2.700 288 39 VLDDENPLTG 2.500 289 303 LTVFLLLVFY 2.500 290 17 DIKPRQPISY 2.500 291 222 FTDMGGNVYI 2.500 292 78 TGEGNFNWRF 2.250 293 113 EAEFRQPAVL 1.800 294 46 LTGEMSSDIY 1.250 295 69 ETDVHFNSLT 1.250 296 47 TGEMSSDIYV 1.125 297 140 SLELQLPDMV 0.900 298 219 DLEFTDMGGN 0.900 299 190 EAEDVEREAQ 0.900 300 244 TVEEAEKRPV 0.900 301 51 SSDIYVKSWV 0.750 302 67 KQETDVHFNS 0.675 303 134 ANDFLGSLEL 0.625 304 120 AVLVLQVWDY 0.500 305 302 LLTVFLLLVF 0.500 306 10 VPAPPPVDIK 0.500 307 95 PTEREVSVWR 0.450 308 241 ELLTVEEAEK 0.400 309 312 YTIPGQISQV 0.250 310 281 KTFVFFIWRR 0.250 311 145 LPDMVRGARG 0.250 312 77 LTGEGNFNWR 0.250 313 12 APPPVDIKPR 0.250 314 154 GPELCSVQLA 0.225 315 216 RPEDLEFTDM 0.225 316 34 NTEDVVLDDE 0.225 317 25 SYELRVVIWN 0.225 318 122 LVLQVWDYDR 0.200 319 231 ILTGKVEAEF 0.200 320 197 EAQEAQAGKK 0.200 321 200 EAQAGKKKRK 0.200 322 100 VSVWRRSGPF 0.150 323 105 RSGPFALEEA 0.150 324 319 SQVIFRPLHK 0.150 325 80 EGNFNWRFVF 0.125 326 293 RTLVLLLLVL 0.125 327 297 LLLLVLLTVF 0.100 328 144 QLPDMVRGAR 0.100 329 242 LLTVEEAEKR 0.100 330 193 DVEREAQEAQ 0.090 331 247 EAEKRPVGKG 0.090 332 62 GLEHDKQETD 0.090 333 245 VEEAEKRPVG 0.090 334 110 ALEEAEFRQP 0.090 335 237 EAEFELLTVE 0.090 336 107 GPFALEEAEF 0.050 337 15 PVDIKPRQPI 0.050 338 304 TVFLLLVFYT 0.050 339 2 WIDIFPQDVP 0.050 340 76 SLTGEGNFNW 0.050 341 307 LLLVFYTIPG 0.050 342 300 LVLLTVFLLL 0.050 343 295 LVLLLLVLLT 0.050 344 301 VLLTVFLLLV 0.050 345 299 LLVLLTVFLL 0.050 346 261 EPLEKPSRPK 0.050 347 277 VNPLKTFVFF 0.050 348 109 FALEEAEFRQ 0.050 349 81 GNFNWRFVFR 0.050 350 296 VLLLLVLLTV 0.050 351 314 IPGQISQVIF 0.050 352 226 GGNVYILTGK 0.050 353 131 RISANDFLGS 0.050 354 97 EREVSVWRRS 0.045 355 239 EFELLTVEEA 0.045 356 111 LEEAEFRQPA 0.045 357 41 DDENPLTGEM 0.045 358 195 EREAQEAQAG 0.045 359 178 RLRGWWPVVK 0.040 360 24 ISYELRVVIW 0.030 361 139 GSLELQLPDM 0.030 362 318 ISQVIFRPLH 0.030 363 224 DMGGNVYILT 0.025 364 165 NGAGPRCNLF 0.025 365 282 TFVFFIWRRY 0.025 366 280 LKTFVFFIWR 0.025 367 82 NFNWRFVFRF 0.025 368 171 CNLFRCRRLR 0.025 369 126 VWDYDRISAN 0.025 370 128 DYDRISANDF 0.025 371 141 LELQLPDMVR 0.025 372 35 TEDVVLDDEN 0.025 373 74 FNSLTGEGNF 0.025 374 221 EFTDMGGNVY 0.025 375 294 TLVLLLLVLL 0.020 376 38 VVLDDENPLT 0.020 377 142 ELQLPDMVRG 0.020 378 53 DIYVKSWVKG 0.020 379 246 EEAEKRPVGK 0.020 380 187 KLKEAEDVER 0.020 381 272 SFNWFVNPLK 0.020 382 298 LLLVLLTVFL 0.020 383 158P3D2 v.2a A1-10mers SEQ. ID Pos 1234567890 Score NO. 101 FSEPQISRGI 13.500 384 138 KADPYVVVSA 10.000 385 170 ILELSISLPA 4.500 386 6 DSDGVNLISM 3.750 387 203 HIDLENRFYS 2.500 388 129 NLAPADPNGK 2.000 389 19 IQDQGEAEVK 1.500 390 199 IGETHIDLEN 1.125 391 93 YPESEAVLFS 1.125 392 108 RGIPQNRPIK 1.000 393 205 DLENRFYSHH 0.900 394 194 GSDDLIGETH 0.750 395 215 RANCGLASQY 0.500 396 99 VLFSEPQISR 0.500 397 180 ETELTVAVFE 0.450 398 117 KLLVRVYVVK 0.400 399 78 GSGHLVGKFK 0.300 400 201 ETHIDLENRF 0.250 401 1 MDDPGDSDGV 0.250 402 73 GGEEEGSGHL 0.225 403 16 VGEIQDQGEA 0.225 404 22 QGEAEVKGTV 0.225 405 167 FGEILELSIS 0.225 406 75 EEEGSGHLVG 0.225 407 36 AVATLKIYNR 0.200 408 30 TVSPKKAVAT 0.200 409 91 LIYPESEAVL 0.200 410 178 PAETELTVAV 0.180 411 24 EAEVKGTVSP 0.180 412 175 ISLPAETELT 0.150 413 45 RSLEEEFNHF 0.150 414 95 ESEAVLFSEP 0.135 415 112 QNRPIKLLVR 0.125 416 54 FEDWLNVFPL 0.125 417 132 PADPNGKADP 0.100 418 81 HLVGKFKGSF 0.100 419 162 QLNPIFGEIL 0.100 420 59 NVFPLYRGQG 0.100 421 142 YVVVSAGRER 0.100 422 227 WVQQGPQEPF 0.100 423 46 SLEEEFNHFE 0.090 424 69 GQDGGGEEEG 0.075 425 140 DPYVVVSAGR 0.050 426 176 SLPAETELTV 0.050 427 197 DLIGETHIDL 0.050 428 35 KAVATLKIYN 0.050 429 29 GTVSPKKAVA 0.050 430 185 VAVFEHDLVG 0.050 431 191 DLVGSDDLIG 0.050 432 109 GIPQNRPIKL 0.050 433 148 GRERQDTKER 0.045 434 74 GEEEGSGHLV 0.045 435 48 EEEFNHFEDW 0.045 436 26 EVKGTVSPKK 0.040 437 221 ASQYEVWVQQ 0.030 438 34 KKAVATLKIY 0.025 439 195 SDDLIGETHI 0.025 440 77 EGSGHLVGKF 0.025 441 56 DWLNVFPLYR 0.025 442 133 ADPNGKADPY 0.025 443 202 THIDLENRFY 0.025 444 127 ATNLAPADPN 0.025 445 183 LTVAVFEHDL 0.025 446 189 EHDLVGSDDL 0.025 447 47 LEEEFNHFED 0.022 448 76 EEGSGHLVGK 0.020 449 186 AVFEHDLVGS 0.020 450 217 NCGLASQYEV 0.020 451 172 ELSISLPAET 0.020 452 97 EAVLFSEPQI 0.020 453 158 YIPKQLNPIF 0.020 454 57 WLNVFPLYRG 0.020 455 146 SAGRERQDTK 0.020 456 18 EIQDQGEAEV 0.020 457 219 GLASQYEVWV 0.020 458 151 RQDTKERYIP 0.015 459 13 ISMVGEIQDQ 0.015 460 145 VSAGRERQDT 0.015 461 211 YSHHRANCGL 0.015 462 84 GKFKGSFLIY 0.013 463 79 SGHLVGKFKG 0.013 464 114 RPIKLLVRVY 0.013 465 164 NPIFGEILEL 0.013 466 8 DGVNLISMVG 0.013 467 103 EPQISRGIPQ 0.013 468 51 FNHFEDWLNV 0.013 469 4 PGDSDGVNLI 0.013 470 179 AETELTVAVF 0.010 471 92 IYPESEAVLF 0.010 472 174 SISLPAETEL 0.010 473 184 TVAVFEHDLV 0.010 474 90 FLIYPESEAV 0.010 475 11 NLISMVGEIQ 0.010 476 98 AVLFSEPQIS 0.010 477 121 RVYVVKATNL 0.010 478 37 VATLKIYNRS 0.010 479 143 VVVSAGRERQ 0.010 480 220 LASQYEVWVQ 0.010 481 130 LAPADPNGKA 0.010 482 105 QISRGIPQNR 0.010 483 158P3D2 v.3 A1-10mers SEQ. ID Pos 1234567890 Score NO. 2 PTEREVSVRR 0.450 484 4 EREVSVRRRS 0.045 485 1 LPTEREVSVR 0.025 486 7 VSVRRRSGPF 0.015 487 6 EVSVRRRSGP 0.001 488 3 TEREVSVRRR 0.001 489 5 REVSVRRRSG 0.001 490 8 SVRRRSGPFA 0.000 491 9 VRRRSGPFAL 0.000 492 10 RRRSGPFALE 0.000 493 158P3D2 v.4 A1-10mers SEQ. ID Pos 1234567890 Score NO. 3 PTEREVSIWR 1.125 494 8 VSIWRRSGPF 0.150 495 5 EREVSIWRRS 0.045 496 1 YLPTEREVSI 0.020 497 2 LPTEREVSIW 0.003 498 7 EVSIWRRSGP 0.001 499 4 TEREVSIWRR 0.001 500 6 REVSIWRRSG 0.001 501 9 SIWRRSGPFA 0.000 502 10 IWRRSGPFAL 0.000 503 158P3D2 v.5a A1-10mers SEQ. ID Pos 1234567890 Score NO. 17 SLDPWSCSYQ 5.000 504 16 TSLDPWSCSY 0.750 505 40 CSWPAMGPGR 0.300 506 45 MGPGRGAICF 0.125 507 6 VWDYTASLPM 0.125 508 29 CVGPGAPSSA 0.100 509 44 AMGPGRGAIC 0.100 510 11 ASLPMTSLDP 0.075 511 36 SSALCSWPAM 0.030 512 15 MTSLDPWSCS 0.025 513 9 YTASLPMTSL 0.025 514 28 WCVGPGAPSS 0.020 515 2 LVLQVWDYTA 0.020 516 37 SALCSWPAMG 0.020 517 21 WSCSYQTWCV 0.015 518 32 PGAPSSALCS 0.013 519 1 VLVLQVWDYT 0.010 520 12 SLPMTSLDPW 0.010 521 39 LCSWPAMGPG 0.010 522 3 VLQVWDYTAS 0.010 523 33 GAPSSALCSW 0.010 524 22 SCSYQTWCVG 0.010 525 49 RGAICFAAAA 0.005 526 38 ALCSWPAMGP 0.005 527 13 LPMTSLDPWS 0.005 528 31 GPGAPSSALC 0.005 529 23 CSYQTWCVGP 0.003 530 4 LQVWDYTASL 0.003 531 25 YQTWCVGPGA 0.003 532 8 DYTASLPMTS 0.003 533 42 WPAMGPGRGA 0.003 534 30 VGPGAPSSAL 0.003 535 35 PSSALCSWPA 0.002 536 18 LDPWSCSYQT 0.001 537 27 TWCVGPGAPS 0.001 538 48 GRGAICFAAA 0.001 539 10 TASLPMTSLD 0.001 540 7 WDYTASLPMT 0.001 541 43 PAMGPGRGAI 0.001 542 24 SYQTWCVGPG 0.001 543 41 SWPAMGPGRG 0.001 544 14 PMTSLDPWSC 0.001 545 46 GPGRGAICFA 0.000 546 26 QTWCVGPGAP 0.000 547 19 DPWSCSYQTW 0.000 548 34 APSSALCSWP 0.000 549 47 PGRGAICFAA 0.000 550 5 QVWDYTASLP 0.000 551 20 PWSCSYQTWC 0.000 552

TABLE VII 158P3D2 A2, 9mers (variants 1, 2a, 3, 4 and 5a) 158P3D2 v.1 A2-9mers SEQ. Pos 123456789 Score ID NO. 302 LLTVFLLLV 1033.404 553 297 LLLLVLLTV 1006.209 554 286 FIWRRYWRT 440.113 555 306 FLLLVFYTI 337.376 556 301 VLLTVFLLL 255.302 557 299 LLVLLTVFL 199.738 558 300 LVLLTVFLL 156.843 559 276 FVNPLKTFV 153.971 560 296 VLLLLVLLT 107.808 561 137 FLGSLELQL 98.267 562 2 WIDIFPQDV 66.867 563 38 VVLDDENPL 48.205 564 48 GEMSSDIYV 27.521 565 31 VIWNTEDVV 27.109 566 295 LVLLLLVLL 27.042 567 313 TIPGQISQV 21.996 568 39 VLDDENPLT 20.776 569 294 TLVLLLLVL 20.145 570 230 YILTGKVEA 11.626 571 144 QLPDMVRGA 9.370 572 293 RTLVLLLLV 8.221 573 30 VVIWNTEDV 5.069 574 141 LELQLPDMV 4.168 575 236 VEAEFELLT 3.838 576 178 RLRGWWPVV 3.684 577 94 LPTEREVSV 3.165 578 180 RGWWPVVKL 2.662 579 228 NVYILTGKV 2.532 580 305 VFLLLVFYT 2.388 581 279 PLKTFVFFI 2.240 582 121 VLVLQVWDY 2.185 583 240 FELLTVEEA 1.853 584 133 SANDFLGSL 1.382 585 124 LQVWDYDRI 1.322 586 224 DMGGNVYIL 1.091 587 118 QPAVLVLQV 1.044 588 46 LTGEMSSDI 1.010 589 83 FNWRFVFRF 0.941 590 27 ELRVVIWNT 0.733 591 140 SLELQLPDM 0.731 592 234 GKVEAEFEL 0.706 593 55 YVKSWVKGL 0.692 594 114 AEFRQPAVL 0.630 595 24 ISYELRVVI 0.623 596 52 SDIYVKSWV 0.531 597 62 GLEHDKQET 0.477 598 177 RRLRGWWPV 0.456 599 22 QPISYELRV 0.454 600 298 LLLVLLTVF 0.442 601 159 SVQLARNGA 0.435 602 76 SLTGEGNFN 0.410 603 235 KVEAEFELL 0.390 604 183 WPVVKLKEA 0.343 605 269 PKTSFNWFV 0.333 606 26 YELRVVIWN 0.312 607 304 TVFLLLVFY 0.305 608 186 VKLKEAEDV 0.298 609 223 TDMGGNVYI 0.295 610 307 LLLVFYTIP 0.219 611 4 DIFPQDVPA 0.190 612 165 NGAGPRCNL 0.139 613 272 SFNWFVNPL 0.130 614 308 LLVFYTIPG 0.127 615 225 MGGNVYILT 0.124 616 10 VPAPPPVDI 0.116 617 112 EEAEFRQPA 0.113 618 135 NDFLGSLEL 0.110 619 143 LQLPDMVRG 0.109 620 281 KTFVFFIWR 0.106 621 171 CNLFRCRRL 0.103 622 8 QDVPAPPPV 0.097 623 318 ISQVIFRPL 0.090 624 87 FVFRFDYLP 0.084 625 86 RFVFRFDYL 0.076 626 93 YLPTEREVS 0.069 627 80 EGNFNWRFV 0.064 628 131 RISANDFLG 0.059 629 290 RYWRTLVLL 0.057 630 314 IPGQISQVI 0.047 631 77 LTGEGNFNW 0.042 632 79 GEGNFNWRF 0.041 633 23 PISYELRVV 0.040 634 70 TDVHFNSLT 0.039 635 109 FALEEAEFR 0.039 636 283 FVFFIWRRY 0.038 637 122 LVLQVWDYD 0.038 638 106 SGPFALEEA 0.037 639 68 QETDVHFNS 0.034 640 168 GPRCNLFRC 0.033 641 292 WRTLVLLLL 0.031 642 245 VEEAEKRPV 0.029 643 319 SQVIFRPLH 0.029 644 231 ILTGKVEAE 0.029 645 317 QISQVIFRP 0.027 646 120 AVLVLQVWD 0.027 647 215 GRPEDLEFT 0.026 648 242 LLTVEEAEK 0.025 649 123 VLQVWDYDR 0.025 650 16 VDIKPRQPI 0.025 651 258 KQPEPLEKP 0.024 652 158P3D2 v.2a A2-9mers SEQ. Pos 123456789 Score ID NO. 117 KLLVRVYVV 849.359 653 91 LIYPESEAV 25.492 654 90 FLIYPESEA 22.853 655 198 LIGETHIDL 20.473 656 158 YIPKQLNPI 15.177 657 220 LASQYEVWV 9.032 658 184 TVAVFEHDL 7.103 659 179 AETELTVAV 5.545 660 19 IQDQGEAEV 4.795 661 176 SLPAETELT 3.651 662 98 AVLFSEPQI 3.378 663 169 EILELSISL 3.342 664 116 IKLLVRVYV 3.342 665 177 LPAETELTV 3.165 666 11 NLISMVGEI 3.119 667 162 QLNPIFGEI 2.577 668 123 YVVKATNLA 2.000 669 218 CGLASQYEV 1.680 670 57 WLNVFPLYR 1.433 671 52 NHFEDWLNV 1.246 672 114 RPIKLLVRV 1.044 673 29 GTVSPKKAV 0.966 674 175 ISLPAETEL 0.877 675 185 VAVFEHDLV 0.805 676 23 GEAEVKGTV 0.721 677 171 LELSISLPA 0.608 678 165 PIFGEILEL 0.550 679 151 RQDTKERYI 0.465 680 191 DLVGSDDLI 0.383 681 84 GKFKGSFLI 0.311 682 161 KQLNPIFGE 0.261 683 55 EDWLNVFPL 0.246 684 137 GKADPYVVV 0.244 685 110 IPQNRPIKL 0.237 686 99 VLFSEPQIS 0.192 687 163 LNPIFGEIL 0.181 688 30 TVSPKKAVA 0.178 689 39 TLKIYNRSL 0.150 690 5 GDSDGVNLI 0.137 691 119 LVRVYVVKA 0.129 692 28 KGTVSPKKA 0.114 693 155 KERYIPKQL 0.110 694 111 PQNRPIKLL 0.110 695 146 SAGRERQDT 0.104 696 204 IDLENRFYS 0.085 697 173 LSISLPAET 0.083 698 31 VSPKKAVAT 0.083 699 8 DGVNLISMV 0.078 700 182 ELTVAVFEH 0.075 701 129 NLAPADPNG 0.075 702 135 PNGKADPYV 0.055 703 34 KKAVATLKI 0.051 704 83 VGKFKGSFL 0.046 705 45 RSLEEEFNH 0.043 706 102 SEPQISRGI 0.041 707 186 AVFEHDLVG 0.041 708 46 SLEEEFNHF 0.037 709 36 AVATLKIYN 0.036 710 112 QNRPIKLLV 0.035 711 222 SQYEVWVQQ 0.034 712 125 VKATNLAPA 0.027 713 14 SMVGEIQDQ 0.025 714 194 GSDDLIGET 0.024 715 105 QISRGIPQN 0.024 716 41 KIYNRSLEE 0.023 717 219 GLASQYEVW 0.022 718 15 MVGEIQDQG 0.022 719 121 RVYVVKATN 0.021 720 167 FGEILELSI 0.020 721 131 APADPNGKA 0.017 722 51 FNHFEDWLN 0.017 723 139 ADPYVVVSA 0.016 724 7 SDGVNLISM 0.016 725 118 LLVRVYVVK 0.016 726 212 SHHRANCGL 0.015 727 74 GEEEGSGHL 0.014 728 206 LENRFYSHH 0.014 729 108 RGIPQNRPI 0.014 730 17 GEIQDQGEA 0.013 731 50 EFNHFEDWL 0.011 732 32 SPKKAVATL 0.011 733 92 IYPESEAVL 0.008 734 61 FPLYRGQGG 0.008 735 22 QGEAEVKGT 0.007 736 136 NGKADPYVV 0.007 737 75 EEEGSGHLV 0.006 738 228 VQQGPQEPF 0.006 739 227 WVQQGPQEP 0.006 740 181 TELTVAVFE 0.006 741 38 ATLKIYNRS 0.006 742 82 LVGKFKGSF 0.005 743 122 VYVVKATNL 0.005 744 81 HLVGKFKGS 0.005 745 86 FKGSFLIYP 0.005 746 192 LVGSDDLIG 0.005 747 120 VRVYVVKAT 0.004 748 196 DDLIGETHI 0.004 749 170 ILELSISLP 0.004 750 2 DDPGDSDGV 0.004 751 35 KAVATLKIY 0.003 752 158P3D2 v.3 A2-9mers Pos 123456789 Score SeqID 9 RRRSGPFAL 0.001 753 8 VRRRSGPFA 0.000 754 4 REVSVRRRS 0.000 755 6 VSVRRRSGP 0.000 756 5 EVSVRRRSG 0.000 757 2 TEREVSVRR 0.000 758 7 SVRRRSGPF 0.000 759 1 PTEREVSVR 0.000 760 3 EREVSVRRR 0.000 761 158P3D2 v.4 A2-9mers SEQ. Pos 123456789 Score ID NO. 1 LPTEREVSI 0.475 762 8 SIWRRSGPF 0.011 763 3 TEREVSIWR 0.000 764 5 REVSIWRRS 0.000 765 9 IWRRSGPFA 0.000 766 7 VSIWRRSGP 0.000 767 6 EVSIWRRSG 0.000 768 2 PTEREVSIW 0.000 769 4 EREVSIWRR 0.000 770 158P3D2 v.5a A2-9mers SEQ. Pos 123456789 Score ID NO. 4 QVWDYTASL 63.609 771 1 LVLQVWDYT 18.791 772 2 VLQVWDYTA 8.446 773 21 SCSYQTWCV 3.405 774 43 AMGPGRGAI 0.980 775 14 MTSLDPWSC 0.880 776 20 WSCSYQTWC 0.820 777 9 TASLPMTSL 0.682 778 25 QTWCVGPGA 0.573 779 36 SALCSWPAM 0.434 780 49 GAICFAAAA 0.262 781 35 SSALCSWPA 0.243 782 30 GPGAPSSAL 0.139 783 6 WDYTASLPM 0.102 784 37 ALCSWPAMG 0.075 785 48 RGAICFAAA 0.062 786 29 VGPGAPSSA 0.055 787 18 DPWSCSYQT 0.030 788 16 SLDPWSCSY 0.030 789 44 MGPGRGAIC 0.023 790 3 LQVWDYTAS 0.019 791 11 SLPMTSLDP 0.015 792 15 TSLDPWSCS 0.013 793 24 YQTWCVGPG 0.010 794 28 CVGPGAPSS 0.007 795 13 PMTSLDPWS 0.007 796 8 YTASLPMTS 0.005 797 47 GRGAICFAA 0.004 798 12 LPMTSLDPW 0.003 799 27 WCVGPGAPS 0.002 800 39 CSWPAMGPG 0.001 801 42 PAMGPGRGA 0.001 802 33 APSSALCSW 0.001 803 22 CSYQTWCVG 0.001 804 32 GAPSSALCS 0.001 805 31 PGAPSSALC 0.001 806 46 PGRGAICFA 0.001 807 45 GPGRGAICF 0.000 808 10 ASLPMTSLD 0.000 809 41 WPAMGPGRG 0.000 810 17 LDPWSCSYQ 0.000 811 7 DYTASLPMT 0.000 812 38 LCSWPAMGP 0.000 813 34 PSSALCSWP 0.000 814 23 SYQTWCVGP 0.000 815 40 SWPAMGPGR 0.000 816 5 VWDYTASLP 0.000 817 19 PWSCSYQTW 0.000 818 26 TWCVGPGAP 0.000 819

TABLE VIII 158P3D2 A2, 10mers (variants 1, 2a, 3, 4 and 5a) 158P3D2 v.1 A2-10mers SEQ. ID Pos 1234567890 Score NO. 301 VLLTVFLLLV 3823.593 820 296 VLLLLVLLTV 1006.209 821 298 LLLVLLTVFL 739.032 822 299 LLVLLTVFLL 484.457 823 93 YLPTEREVSV 319.939 824 304 TVFLLLVFYT 177.011 825 278 NPLKTFVFFI 70.254 826 294 TLVLLLLVLL 49.134 827 26 YELRVVIWNT 42.542 828 286 FIWRRYWRTL 38.130 829 300 LVLLTVFLLL 22.339 830 236 VEAEFELLTV 21.680 831 101 SVWRRSGPFA 19.844 832 31 VIWNTEDVVL 16.993 833 38 VVLDDENPLT 16.816 834 87 FVFRFDYLPT 16.647 835 117 RQPAVLVLQV 16.219 836 125 QVWDYDRISA 14.793 837 123 VLQVWDYDRI 13.036 838 312 YTIPGQISQV 10.220 839 295 LVLLLLVLLT 9.433 840 63 LEHDKQETDV 9.426 841 21 RQPISYELRV 7.052 842 114 AEFRQPAVLV 5.004 843 271 TSFNWFVNPL 4.510 844 68 QETDVHFNSL 3.236 845 29 RVVIWNTEDV 2.982 846 61 KGLEHDKQET 2.583 847 79 GEGNFNWRFV 2.529 848 268 RPKTSFNWFV 2.491 849 140 SLELQLPDMV 2.181 850 30 VVIWNTEDVV 2.078 851 273 FNWFVNPLKT 1.857 852 222 FTDMGGNVYI 1.466 853 143 LQLPDMVRGA 1.457 854 275 WFVNPLKTFV 1.222 855 139 GSLELQLPDM 1.132 856 317 QISQVIFRPL 1.116 857 220 LEFTDMGGNV 1.106 858 293 RTLVLLLLVL 1.035 859 51 SSDIYVKSWV 0.999 860 309 LVFYTIPGQI 0.746 861 224 DMGGNVYILT 0.605 862 306 FLLLVFYTIP 0.593 863 313 TIPGQISQVI 0.588 864 153 RGPELCSVQL 0.572 865 235 KVEAEFELLT 0.555 866 307 LLLVFYTIPG 0.469 867 297 LLLLVLLTVF 0.442 868 167 AGPRCNLFRC 0.433 869 76 SLTGEGNFNW 0.432 870 120 AVLVLQVWDY 0.416 871 112 EEAEFRQPAV 0.416 872 244 TVEEAEKRPV 0.319 873 91 FDYLPTEREV 0.284 874 189 KEAEDVEREA 0.277 875 172 NLFRCRRLRG 0.276 876 132 ISANDFLGSL 0.269 877 285 FFIWRRYWRT 0.268 878 85 WRFVFRFDYL 0.259 879 1 MWIDIFPQDV 0.256 880 148 MVRGARGPEL 0.242 881 45 PLTGEMSSDI 0.230 882 39 VLDDENPLTG 0.208 883 185 VVKLKEAEDV 0.177 884 281 KTFVFFIWRR 0.176 885 151 GARGPELCSV 0.169 886 47 TGEMSSDIYV 0.160 887 137 FLGSLELQLP 0.158 888 37 DVVLDDENPL 0.140 889 164 RNGAGPRCNL 0.139 890 231 ILTGKVEAEF 0.127 891 283 FVFFIWRRYW 0.122 892 302 LLTVFLLLVF 0.119 893 121 VLVLQVWDYD 0.116 894 234 GKVEAEFELL 0.113 895 258 KQPEPLEKPS 0.108 896 223 TDMGGNVYIL 0.104 897 292 WRTLVLLLLV 0.102 898 305 VFLLLVFYTI 0.087 899 22 QPISYELRVV 0.086 900 109 FALEEAEFRQ 0.084 901 214 KGRPEDLEFT 0.080 902 276 FVNPLKTFVF 0.071 903 9 DVPAPPPVDI 0.068 904 7 PQDVPAPPPV 0.062 905 227 GNVYILTGKV 0.059 906 308 LLVFYTIPGQ 0.058 907 290 RYWRTLVLLL 0.057 908 134 ANDFLGSLEL 0.056 909 194 VEREAQEAQA 0.051 910 111 LEEAEFRQPA 0.040 911 230 YILTGKVEAE 0.039 912 19 KPRQPISYEL 0.037 913 105 RSGPFALEEA 0.037 914 158 CSVQLARNGA 0.032 915 233 TGKVEAEFEL 0.028 916 129 YDRISANDFL 0.028 917 170 RCNLFRCRRL 0.028 918 177 RRLRGWWPVV 0.025 919 158P3D2 v.2a A2-10mers SEQ. ID Pos 1234567890 Score NO. 219 GLASQYEVWV 382.536 920 90 FLIYPESEAV 156.770 921 176 SLPAETELTV 69.552 922 118 LLVRVYVVKA 19.425 923 82 LVGKFKGSFL 17.477 924 162 QLNPIFGEIL 16.308 925 54 FEDWLNVFPL 10.196 926 91 LIYPESEAVL 6.551 927 121 RVYVVKATNL 5.981 928 51 FNHFEDWLNV 3.550 929 161 KQLNPIFGEI 3.383 930 184 TVAVFEHDLV 2.982 931 18 EIQDQGEAEV 2.941 932 174 SISLPAETEL 2.937 933 109 GIPQNRPIKL 2.937 934 183 LTVAVFEHDL 1.917 935 197 DLIGETHIDL 1.602 936 28 KGTVSPKKAV 1.589 937 49 EEFNHFEDWL 1.180 938 57 WLNVFPLYRG 0.788 939 30 TVSPKKAVAT 0.652 940 211 YSHHRANCGL 0.641 941 116 IKLLVRVYVV 0.573 942 172 ELSISLPAET 0.559 943 31 VSPKKAVATL 0.545 944 110 IPQNRPIKLL 0.545 945 170 ILELSISLPA 0.541 946 21 DQGEAEVKGT 0.534 947 217 NCGLASQYEV 0.454 948 168 GEILELSISL 0.415 949 74 GEEEGSGHLV 0.355 950 164 NPIFGEILEL 0.321 951 222 SQYEVWVQQG 0.228 952 186 AVFEHDLVGS 0.228 953 138 KADPYVVVSA 0.222 954 7 SDGVNLISMV 0.222 955 38 ATLKIYNRSL 0.220 956 177 LPAETELTVA 0.213 957 119 LVRVYVVKAT 0.194 958 134 DPNGKADPYV 0.187 959 111 PQNRPIKLLV 0.155 960 175 ISLPAETELT 0.150 961 10 VNLISMVGEI 0.128 962 117 KLLVRVYVVK 0.119 963 193 VGSDDLIGET 0.101 964 99 VLFSEPQISR 0.094 965 145 VSAGRERQDT 0.083 966 46 SLEEEFNHFE 0.082 967 181 TELTVAVFEH 0.072 968 124 VVKATNLAPA 0.059 969 166 IFGEILELSI 0.050 970 3 DPGDSDGVNL 0.043 971 115 PIKLLVRVYV 0.041 972 1 MDDPGDSDGV 0.032 973 29 GTVSPKKAVA 0.028 974 14 SMVGEIQDQG 0.026 975 41 KIYNRSLEEE 0.026 976 83 VGKFKGSFLI 0.024 977 113 NRPIKLLVRV 0.022 978 35 KAVATLKIYN 0.020 979 158 YIPKQLNPIF 0.019 980 198 LIGETHIDLE 0.016 981 130 LAPADPNGKA 0.015 982 129 NLAPADPNGK 0.015 983 227 WVQQGPQEPF 0.015 984 45 RSLEEEFNHF 0.014 985 89 SFLIYPESEA 0.013 986 27 VKGTVSPKKA 0.012 987 209 RFYSHHRANC 0.011 988 97 EAVLFSEPQI 0.011 989 98 AVLFSEPQIS 0.010 990 136 NGKADPYVVV 0.010 991 15 MVGEIQDQGE 0.009 992 123 YVVKATNLAP 0.006 993 195 SDDLIGETHI 0.006 994 179 AETELTVAVF 0.006 995 188 FEHDLVGSDD 0.005 996 169 EILELSISLP 0.005 997 192 LVGSDDLIGE 0.005 998 204 IDLENRFYSH 0.005 999 73 GGEEEGSGHL 0.005 1000 203 HIDLENRFYS 0.004 1001 171 LELSISLPAE 0.004 1002 135 PNGKADPYVV 0.004 1003 101 FSEPQISRGI 0.004 1004 22 QGEAEVKGTV 0.004 1005 12 LISMVGEIQD 0.003 1006 157 RYIPKQLNPI 0.003 1007 59 NVFPLYRGQG 0.003 1008 9 GVNLISMVGE 0.003 1009 36 AVATLKIYNR 0.003 1010 11 NLISMVGEIQ 0.003 1011 79 SGHLVGKFKG 0.003 1012 37 VATLKIYNRS 0.003 1013 87 KGSFLIYPES 0.003 1014 220 LASQYEVWVQ 0.002 1015 23 GEAEVKGTVS 0.002 1016 191 DLVGSDDLIG 0.002 1017 6 DSDGVNLISM 0.002 1018 105 QISRGIPQNR 0.002 1019 158P3D2 v.3 A2-10mers SEQ. ID Pos 1234567890 Score NO. 8 SVRRRSGPFA 0.182 1020 9 VRRRSGPFAL 0.002 1021 1 LPTEREVSVR 0.001 1022 5 REVSVRRRSG 0.000 1023 7 VSVRRRSGPF 0.000 1024 6 EVSVRRRSGP 0.000 1025 3 TEREVSVRRR 0.000 1026 10 RRRSGPFALE 0.000 1027 2 PTEREVSVRR 0.000 1028 4 EREVSVRRRS 0.000 1029 158P3D2 v.4 A2-10mers SEQ. ID Pos 1234567890 Score NO. 1 YLPTEREVSI 47.991 1030 9 SIWRRSGPFA 31.184 1031 2 LPTEREVSIW 0.003 1032 10 IWRRSGPFAL 0.002 1033 4 TEREVSIWRR 0.002 1034 6 REVSIWRRSG 0.000 1035 8 VSIWRRSGPF 0.000 1036 7 EVSIWRRSGP 0.000 1037 3 PTEREVSIWR 0.000 1038 5 EREVSIWRRS 0.000 1039 158P3D2 v.5a A2-10mers SEQ. ID Pos 1234567890 Score NO. 1 VLVLQVWDYT 58.040 1040 21 WSCSYQTWCV 15.664 1041 4 LQVWDYTASL 3.682 1042 9 YTASLPMTSL 3.139 1043 2 LVLQVWDYTA 2.734 1044 25 YQTWCVGPGA 2.317 1045 44 AMGPGRGAIC 1.471 1046 14 PMTSLDPWSC 0.592 1047 29 CVGPGAPSSA 0.435 1048 46 GPGRGAICFA 0.410 1049 7 WDYTASLPMT 0.350 1050 30 VGPGAPSSAL 0.237 1051 3 VLQVWDYTAS 0.190 1052 49 RGAICFAAAA 0.123 1053 12 SLPMTSLDPW 0.084 1054 36 SSALCSWPAM 0.055 1055 5 QVWDYTASLP 0.044 1056 17 SLDPWSCSYQ 0.033 1057 31 GPGAPSSALC 0.032 1058 42 WPAMGPGRGA 0.030 1059 18 LDPWSCSYQT 0.018 1060 13 LPMTSLDPWS 0.017 1061 38 ALCSWPAMGP 0.015 1062 16 TSLDPWSCSY 0.007 1063 35 PSSALCSWPA 0.005 1064 37 SALCSWPAMG 0.004 1065 15 MTSLDPWSCS 0.003 1066 33 GAPSSALCSW 0.002 1067 28 WCVGPGAPSS 0.002 1068 43 PAMGPGRGAI 0.002 1069 48 GRGAICFAAA 0.001 1070 45 MGPGRGAICF 0.001 1071 40 CSWPAMGPGR 0.001 1072 34 APSSALCSWP 0.001 1073 6 VWDYTASLPM 0.000 1074 19 DPWSCSYQTW 0.000 1075 11 ASLPMTSLDP 0.000 1076 22 SCSYQTWCVG 0.000 1077 47 PGRGAICFAA 0.000 1078 23 CSYQTWCVGP 0.000 1079 39 LCSWPAMGPG 0.000 1080 26 QTWCVGPGAP 0.000 1081 10 TASLPMTSLD 0.000 1082 20 PWSCSYQTWC 0.000 1083 32 PGAPSSALCS 0.000 1084 27 TWCVGPGAPS 0.000 1085 24 SYQTWCVGPG 0.000 1086 41 SWPAMGPGRG 0.000 1087 8 DYTASLPMTS 0.000 1088

TABLE IX 158P3D2 A3, 9mers (variants 1, 2a, 3, 4 and 5a) 158P3D2 v.1 A3-9mers SEQ. Pos 123456789 Score ID NO. 281 KTFVFFIWR 54.000 1089 121 VLVLQVWDY 54.000 1090 123 VLQVWDYDR 36.000 1091 49 EMSSDIYVK 27.000 1092 242 LLTVEEAEK 20.000 1093 306 FLLLVFYTI 12.150 1094 53 DIYVKSWVK 9.000 1095 301 VLLTVFLLL 8.100 1096 320 QVIFRPLHK 6.000 1097 298 LLLVLLTVF 4.500 1098 142 ELQLPDMVR 3.600 1099 156 ELCSVQLAR 3.600 1100 316 GQISQVIFR 3.240 1101 59 WVKGLEHDK 3.000 1102 304 TVFLLLVFY 3.000 1103 294 TLVLLLLVL 2.700 1104 224 DMGGNVYIL 2.430 1105 172 NLFRCRRLR 2.000 1106 302 LLTVFLLLV 1.800 1107 279 PLKTFVFFI 1.620 1108 297 LLLLVLLTV 1.350 1109 137 FLGSLELQL 1.200 1110 181 GWWPVVKLK 1.013 1111 299 LLVLLTVFL 0.900 1112 296 VLLLLVLLT 0.900 1113 178 RLRGWWPVV 0.900 1114 300 LVLLTVFLL 0.810 1115 81 GNFNWRFVF 0.540 1116 235 KVEAEFELL 0.540 1117 83 FNWRFVFRF 0.540 1118 303 LTVFLLLVF 0.450 1119 243 LTVEEAEKR 0.450 1120 201 AQAGKKKRK 0.450 1121 227 GNVYILTGK 0.405 1122 62 GLEHDKQET 0.300 1123 273 FNWFVNPLK 0.300 1124 262 PLEKPSRPK 0.300 1125 283 FVFFIWRRY 0.300 1126 101 SVWRRSGPF 0.300 1127 140 SLELQLPDM 0.300 1128 55 YVKSWVKGL 0.270 1129 27 ELRVVIWNT 0.203 1130 222 FTDMGGNVY 0.200 1131 85 WRFVFRFDY 0.180 1132 308 LLVFYTIPG 0.180 1133 198 AQEAQAGKK 0.180 1134 79 GEGNFNWRF 0.162 1135 286 FIWRRYWRT 0.150 1136 232 LTGKVEAEF 0.150 1137 295 LVLLLLVLL 0.135 1138 11 PAPPPVDIK 0.135 1139 21 RQPISYELR 0.120 1140 170 RGNLFRCRR 0.120 1141 31 VIWNTEDVV 0.100 1142 39 VLDDENPLT 0.100 1143 278 NPLKTFVFF 0.090 1144 187 KLKEAEDVE 0.090 1145 231 ILTGKVEAE 0.090 1146 265 KPSRPKTSF 0.090 1147 87 FVFRFDYLP 0.090 1148 110 ALEEAEFRQ 0.090 1149 307 LLLVFYTIP 0.090 1150 38 VVLDDENPL 0.090 1151 166 GAGPRCNLF 0.090 1152 109 FALEEAEFR 0.090 1153 197 EAQEAQAGK 0.090 1154 282 TFVFFIWRR 0.081 1155 179 LRGWWPVVK 0.060 1156 257 RKQPEPLEK 0.060 1157 144 QLPDMVRGA 0.060 1158 268 RPKTSFNWF 0.060 1159 247 EAEKRPVGK 0.060 1160 2 WIDIFPQDV 0.060 1161 46 LTGEMSSDI 0.045 1162 293 RTLVLLLLV 0.045 1163 4 DIFPQDVPA 0.045 1164 77 LTGEGNFNW 0.045 1165 313 TIPGQISQV 0.045 1166 93 YLPTEREVS 0.040 1167 230 YILTGKVEA 0.030 1168 76 SLTGEGNFN 0.030 1169 228 NVYILTGKV 0.030 1170 57 KSWVKGLEH 0.030 1171 276 FVNPLKTFV 0.030 1172 30 VVIWNTEDV 0.030 1173 199 QEAQAGKKK 0.030 1174 69 ETDVHFNSL 0.027 1175 319 SQVIFRPLH 0.027 1176 168 GPRCNLFRC 0.027 1177 124 LQVWDYDRI 0.027 1178 96 TEREVSVWR 0.027 1179 24 ISYELRVVI 0.022 1180 159 SVQLARNGA 0.020 1181 161 QLARNGAGP 0.020 1182 285 FFIWRRYWR 0.018 1183 250 KRPVGKGRK 0.018 1184 214 KGRPEDLEF 0.018 1185 78 TGEGNFNWR 0.018 1186 154 GPELCSVQL 0.018 1187 22 QPISYELRV 0.018 1188 158P3D2 v.2a A3-9mers SEQ. Pos 123456789 Score ID NO. 118 LLVRVYVVK 45.000 1189 57 WLNVFPLYR 24.000 1190 46 SLEEEFNHF 9.000 1191 117 KLLVRVYVV 8.100 1192 109 GIPQNRPIK 6.000 1193 26 EVKGTVSPK 2.700 1194 162 QLNPIFGEI 1.215 1195 153 DTKERYIPK 0.900 1196 11 NLISMVGEI 0.810 1197 219 GLASQYEVW 0.600 1198 182 ELTVAVFEH 0.540 1199 205 DLENRFYSH 0.540 1200 90 FLIYPESEA 0.450 1201 191 DLVGSDDLI 0.405 1202 130 LAPADPNGK 0.200 1203 99 VLFSEPQIS 0.200 1204 37 VATLKIYNR 0.180 1205 184 TVAVFEHDL 0.180 1206 82 LVGKFKGSF 0.180 1207 39 TLKIYNRSL 0.180 1208 119 LVRVYVVKA 0.180 1209 198 LIGETHIDL 0.180 1210 91 LIYPESEAV 0.150 1211 165 PIFGEILEL 0.135 1212 228 VQQGPQEPF 0.135 1213 81 HLVGKFKGS 0.135 1214 35 KAVATLKIY 0.135 1215 85 KFKGSFLIY 0.108 1216 176 SLPAETELT 0.100 1217 201 ETHIDLENR 0.090 1218 98 AVLFSEPQI 0.090 1219 180 ETELTVAVF 0.090 1220 158 YIPKQLNPI 0.090 1221 169 EILELSISL 0.081 1222 14 SMVGEIQDQ 0.068 1223 143 VVVSAGRER 0.060 1224 41 KIYNRSLEE 0.060 1225 106 ISRGIPQNR 0.045 1226 203 HIDLENRFY 0.040 1227 29 GTVSPKKAV 0.034 1228 20 QDQGEAEVK 0.030 1229 27 VKGTVSPKK 0.030 1230 147 AGRERQDTK 0.030 1231 123 YVVKATNLA 0.030 1232 129 NLAPADPNG 0.030 1233 170 ILELSISLP 0.030 1234 186 AVFEHDLVG 0.030 1235 30 TVSPKKAVA 0.030 1236 84 GKFKGSFLI 0.027 1237 78 GSGHLVGKF 0.027 1238 93 YPESEAVLF 0.020 1239 159 IPKQLNPIF 0.020 1240 161 KQLNPIFGE 0.018 1241 100 LFSEPQISR 0.018 1242 9 GVNLISMVG 0.018 1243 32 SPKKAVATL 0.018 1244 134 DPNGKADPY 0.018 1245 138 KADPYVVVS 0.016 1246 79 SGHLVGKFK 0.015 1247 121 RVYVVKATN 0.015 1248 197 DLIGETHID 0.013 1249 77 EGSGHLVGK 0.013 1250 115 PIKLLVRVY 0.012 1251 216 ANCGLASQY 0.012 1252 113 NRPIKLLVR 0.012 1253 110 IPQNRPIKL 0.012 1254 53 HFEDWLNVF 0.009 1255 172 ELSISLPAE 0.009 1256 56 DWLNVFPLY 0.008 1257 55 EDWLNVFPL 0.008 1258 207 ENRFYSHHR 0.007 1259 45 RSLEEEFNH 0.007 1260 175 ISLPAETEL 0.007 1261 222 SQYEVWVQQ 0.007 1262 183 LTVAVFEHD 0.007 1263 149 RERQDTKER 0.006 1264 220 LASQYEVWV 0.006 1265 19 IQDQGEAEV 0.006 1266 177 LPAETELTV 0.006 1267 5 GDSDGVNLI 0.005 1268 114 RPIKLLVRV 0.005 1269 38 ATLKIYNRS 0.005 1270 15 MVGEIQDQG 0.005 1271 88 GSFLIYPES 0.005 1272 155 KERYIPKQL 0.004 1273 36 AVATLKIYN 0.004 1274 43 YNRSLEEEF 0.004 1275 124 VVKATNLAP 0.004 1276 192 LVGSDDLIG 0.004 1277 34 KKAVATLKI 0.004 1278 163 LNPIFGEIL 0.004 1279 202 THIDLENRF 0.003 1280 33 PKKAVATLK 0.003 1281 185 VAVFEHDLV 0.003 1282 52 NHFEDWLNV 0.003 1283 105 QISRGIPQN 0.003 1284 12 LISMVGEIQ 0.003 1285 62 PLYRGQGGQ 0.003 1286 174 SISLPAETE 0.003 1287 69 GQDGGGEEE 0.003 1288 158P3D2 v.3 A3-9mers SEQ. Pos 123456789 Score ID NO. 1 PTEREVSVR 0.060 1289 7 SVRRRSGPF 0.060 1290 2 TEREVSVRR 0.027 1291 9 RRRSGPFAL 0.002 1292 3 EREVSVRRR 0.000 1293 8 VRRRSGPFA 0.000 1294 6 VSVRRRSGP 0.000 1295 5 EVSVRRRSG 0.000 1296 4 REVSVRRRS 0.000 1297 158P3D2 v.4 A3-9mers SEQ. Pos 123456789 Score ID NO. 8 SIWRRSGPF 0.300 1298 3 TEREVSIWR 0.054 1299 1 LPTEREVSI 0.009 1300 4 EREVSIWRR 0.005 1301 2 PTEREVSIW 0.003 1302 9 IWRRSGPFA 0.000 1303 6 EVSIWRRSG 0.000 1304 7 VSIWRRSGP 0.000 1305 5 REVSIWRRS 0.000 1306 158P3D2 v.5a A3-9mers SEQ. Pos 123456789 Score ID NO. 16 SLDPWSCSY 18.000 1307 2 VLQVWDYTA 1.800 1308 4 QVWDYTASL 0.900 1309 43 AMGPGRGAI 0.270 1310 45 GPGRGAICF 0.120 1311 25 QTWCVGPGA 0.075 1312 37 ALCSWPAMG 0.060 1313 11 SLPMTSLDP 0.040 1314 14 MTSLDPWSC 0.030 1315 30 GPGAPSSAL 0.027 1316 49 GAICFAAAA 0.027 1317 1 LVLQVWDYT 0.022 1318 9 TASLPMTSL 0.013 1319 21 SCSYQTWCV 0.006 1320 28 CVGPGAPSS 0.006 1321 12 LPMTSLDPW 0.005 1322 18 DPWSCSYQT 0.005 1323 8 YTASLPMTS 0.004 1324 13 PMTSLDPWS 0.004 1325 40 SWPAMGPGR 0.004 1326 33 APSSALCSW 0.003 1327 20 WSCSYQTWC 0.003 1328 35 SSALCSWPA 0.003 1329 36 SALCSWPAM 0.003 1330 47 GRGAICFAA 0.003 1331 32 GAPSSALCS 0.002 1332 6 WDYTASLPM 0.002 1333 3 LQVWDYTAS 0.002 1334 27 WCVGPGAPS 0.001 1335 38 LCSWPAMGP 0.001 1336 48 RGAICFAAA 0.001 1337 24 YQTWCVGPG 0.001 1338 22 CSYQTWCVG 0.001 1339 15 TSLDPWSCS 0.000 1340 39 CSWPAMGPG 0.000 1341 29 VGPGAPSSA 0.000 1342 44 MGPGRGAIC 0.000 1343 10 ASLPMTSLD 0.000 1344 42 PAMGPGRGA 0.000 1345 23 SYQTWCVGP 0.000 1346 41 WPAMGPGRG 0.000 1347 46 PGRGAICFA 0.000 1348 7 DYTASLPMT 0.000 1349 31 PGAPSSALC 0.000 1350 5 VWDYTASLP 0.000 1351 17 LDPWSCSYQ 0.000 1352 19 PWSCSYQTW 0.000 1353 34 PSSALCSWP 0.000 1354 26 TWCVGPGAP 0.000 1355

TABLE X 158P3D2 A3, 10mers (variants 1, 2a, 3, 4 and 5a) 158P3D2 v.1 A3-10mers SEQ. Pos 1234567890 Score ID NO. 178 RLRGWWPVVK 90.000 1356 281 KTFVFFIWRR 40.500 1357 187 KLKEAEDVER 18.000 1358 241 ELLTVEEAEK 9.000 1359 299 LLVLLTVFLL 8.100 1360 302 LLTVFLLLVF 6.000 1361 122 LVLQVWDYDR 5.400 1362 120 AVLVLQVWDY 5.400 1363 297 LLLLVLLTVF 4.500 1364 231 ILTGKVEAEF 4.500 1365 242 LLTVEEAEKR 4.000 1366 301 VLLTVFLLLV 2.700 1367 144 QLPDMVRGAR 1.800 1368 319 SQVIFRPLHK 1.800 1369 296 VLLLLVLLTV 1.350 1370 294 TLVLLLLVLL 1.350 1371 10 VPAPPPVDIK 1.350 1372 48 GEMSSDIYVK 1.215 1373 161 QLARNGAGPR 1.200 1374 298 LLLVLLTVFL 0.900 1375 77 LTGEGNFNWR 0.900 1376 276 FVNPLKTFVF 0.900 1377 76 SLTGEGNFNW 0.900 1378 300 LVLLTVFLLL 0.810 1379 123 VLQVWDYDRI 0.600 1380 303 LTVFLLLVFY 0.450 1381 304 TVFLLLVFYT 0.450 1382 81 GNFNWRFVFR 0.360 1383 17 DIKPRQPISY 0.360 1384 166 GAGPRCNLFR 0.360 1385 31 VIWNTEDVVL 0.300 1386 107 GPFALEEAEF 0.300 1387 46 LTGEMSSDIY 0.300 1388 198 AQEAQAGKKK 0.300 1389 279 PLKTFVFFIW 0.270 1390 278 NPLKTFVFFI 0.243 1391 180 RGWWPVVKLK 0.225 1392 93 YLPTEREVSV 0.200 1393 140 SLELQLPDMV 0.200 1394 172 NLFRCRRLRG 0.200 1395 125 QVWDYDRISA 0.200 1396 307 LLLVFYTIPG 0.180 1397 235 KVEAEFELLT 0.180 1398 96 TEREVSVWRR 0.162 1399 226 GGNVYILTGK 0.135 1400 293 RTLVLLLLVL 0.135 1401 309 LVFYTIPGQI 0.135 1402 224 DMGGNVYILT 0.135 1403 271 TSFNWFVNPL 0.135 1404 313 TIPGQISQVI 0.135 1405 256 GRKQPEPLEK 0.120 1406 87 FVFRFDYLPT 0.100 1407 101 SVWRRSGPFA 0.100 1408 52 SDIYVKSWVK 0.090 1409 295 LVLLLLVLLT 0.090 1410 148 MVRGARGPEL 0.090 1411 306 FLLLVFYTIP 0.090 1412 45 PLTGEMSSDI 0.090 1413 286 FIWRRYWRTL 0.090 1414 19 KPRQPISYEL 0.081 1415 280 LKTFVFFIWR 0.072 1416 62 GLEHDKQETD 0.060 1417 259 QPEPLEKPSR 0.060 1418 284 VFFIWRRYWR 0.060 1419 196 REAQEAQAGK 0.060 1420 82 NFNWRFVFRF 0.054 1421 141 LELQLPDMVR 0.054 1422 121 VLVLQVWDYD 0.045 1423 308 LLVFYTIPGQ 0.045 1424 12 APPPVDIKPR 0.045 1425 39 VLDDENPLTG 0.040 1426 84 NWRFVFRFDY 0.036 1427 168 GPRCNLFRCR 0.036 1428 117 RQPAVLVLQV 0.036 1429 21 RQPISYELRV 0.036 1430 312 YTIPGQISQV 0.034 1431 272 SFNWFVNPLK 0.030 1432 58 SWVKGLEHDK 0.030 1433 200 EAQAGKKKRK 0.030 1434 30 VVIWNTEDVV 0.030 1435 283 FVFFIWRRYW 0.030 1436 137 FLGSLELQLP 0.030 1437 222 FTDMGGNVYI 0.030 1438 29 RVVIWNTEDV 0.030 1439 95 PTEREVSVWR 0.030 1440 37 DVVLDDENPL 0.027 1441 78 TGEGNFNWRF 0.027 1442 9 DVPAPPPVDI 0.027 1443 317 QISQVIFRPL 0.027 1444 270 KTSFNWFVNP 0.027 1445 246 EEAEKRPVGK 0.027 1446 197 EAQEAQAGKK 0.027 1447 131 RISANDFLGS 0.024 1448 24 ISYELRVVIW 0.022 1449 261 EPLEKPSRPK 0.020 1450 314 IPGQISQVIF 0.020 1451 202 QAGKKKRKQR 0.020 1452 89 FRFDYLPTER 0.020 1453 185 VVKLKEAEDV 0.020 1454 316 GQISQVIFRP 0.018 1455 158P3D2 v.2a A3-10mers SEQ. Pos 1234567890 Score ID NO. 117 KLLVRVYVVK 135.000 1456 99 VLFSEPQISR 60.000 1457 129 NLAPADPNGK 30.000 1458 81 HLVGKFKGSF 4.050 1459 162 QLNPIFGEIL 2.700 1460 118 LLVRVYVVKA 2.700 1461 219 GLASQYEVWV 1.800 1462 36 AVATLKIYNR 1.800 1463 26 EVKGTVSPKK 1.350 1464 197 DLIGETHIDL 0.810 1465 170 ILELSISLPA 0.600 1466 105 QISRGIPQNR 0.600 1467 19 IQDQGEAEVK 0.600 1468 91 LIYPESEAVL 0.450 1469 176 SLPAETELTV 0.400 1470 84 GKFKGSFLIY 0.360 1471 109 GIPQNRPIKL 0.360 1472 90 FLIYPESEAV 0.300 1473 121 RVYVVKATNL 0.300 1474 32 SPKKAVATLK 0.300 1475 227 WVQQGPQEPF 0.300 1476 25 AEVKGTVSPK 0.270 1477 78 GSGHLVGKFK 0.225 1478 158 YIPKQLNPIF 0.200 1479 146 SAGRERQDTK 0.200 1480 205 DLENRFYSHH 0.180 1481 183 LTVAVFEHDL 0.135 1482 57 WLNVFPLYRG 0.135 1483 161 KQLNPIFGEI 0.109 1484 46 SLEEEFNHFE 0.090 1485 140 DPYVVVSAGR 0.090 1486 45 RSLEEEFNHF 0.068 1487 52 NHFEDWLNVF 0.068 1488 14 SMVGEIQDQG 0.068 1489 142 YVVVSAGRER 0.060 1490 82 LVGKFKGSFL 0.060 1491 174 SISLPAETEL 0.060 1492 200 GETHIDLENR 0.054 1493 108 RGIPQNRPIK 0.045 1494 41 KIYNRSLEEE 0.045 1495 186 AVFEHDLVGS 0.045 1496 11 NLISMVGEIQ 0.045 1497 29 GTVSPKKAVA 0.045 1498 222 SQYEVWVQQG 0.041 1499 138 KADPYVVVSA 0.041 1500 215 RANCGLASQY 0.040 1501 152 QDTKERYIPK 0.040 1502 112 QNRPIKLLVR 0.036 1503 206 LENRFYSHHR 0.036 1504 172 ELSISLPAET 0.030 1505 201 ETHIDLENRF 0.030 1506 124 VVKATNLAPA 0.030 1507 182 ELTVAVFEHD 0.027 1508 164 NPIFGEILEL 0.027 1509 76 EEGSGHLVGK 0.027 1510 191 DLVGSDDLIG 0.027 1511 55 EDWLNVFPLY 0.027 1512 179 AETELTVAVF 0.027 1513 119 LVRVYVVKAT 0.022 1514 39 TLKIYNRSLE 0.020 1515 184 TVAVFEHDLV 0.020 1516 114 RPIKLLVRVY 0.018 1517 168 GEILELSISL 0.016 1518 54 FEDWLNVFPL 0.016 1519 30 TVSPKKAVAT 0.015 1520 38 ATLKIYNRSL 0.013 1521 59 NVFPLYRGQG 0.013 1522 203 HIDLENRFYS 0.012 1523 149 RERQDTKERY 0.012 1524 56 DWLNVFPLYR 0.011 1525 34 KKAVATLKIY 0.009 1526 31 VSPKKAVATL 0.009 1527 9 GVNLISMVGE 0.009 1528 181 TELTVAVFEH 0.008 1529 49 EEFNHFEDWL 0.008 1530 165 PIFGEILELS 0.007 1531 110 IPQNRPIKLL 0.007 1532 148 GRERQDTKER 0.006 1533 123 YVVKATNLAP 0.006 1534 192 LVGSDDLIGE 0.006 1535 217 NCGLASQYEV 0.006 1536 98 AVLFSEPQIS 0.006 1537 18 EIQDQGEAEV 0.006 1538 88 GSFLIYPESE 0.005 1539 198 LIGETHIDLE 0.005 1540 177 LPAETELTVA 0.005 1541 194 GSDDLIGETH 0.005 1542 204 IDLENRFYSH 0.004 1543 12 LISMVGEIQD 0.004 1544 133 ADPNGKADPY 0.004 1545 92 IYPESEAVLF 0.003 1546 15 MVGEIQDQGE 0.003 1547 225 EVWVQQGPQE 0.003 1548 115 PIKLLVRVYV 0.003 1549 62 PLYRGQGGQD 0.003 1550 211 YSHHRANCGL 0.003 1551 143 VVVSAGRERQ 0.003 1552 116 IKLLVRVYVV 0.003 1553 69 GQDGGGEEEG 0.003 1554 97 EAVLFSEPQI 0.003 1555 158P3D2 v.3 A3-10mers SEQ. Pos 1234567890 Score ID NO. 1 LPTEREVSVR 0.180 1556 2 PTEREVSVRR 0.030 1557 8 SVRRRSGPFA 0.020 1558 3 TEREVSVRRR 0.005 1559 7 VSVRRRSGPF 0.005 1560 9 VRRRSGPFAL 0.002 1561 6 EVSVRRRSGP 0.001 1562 10 RRRSGPFALE 0.000 1563 5 REVSVRRRSG 0.000 1564 4 EREVSVRRRS 0.000 1565 158P3D2 v.4 A3-10mers SEQ. Pos 1234567890 Score ID NO. 1 YLPTEREVSI 0.600 1566 9 SIWRRSGPFA 0.100 1567 4 TEREVSIWRR 0.081 1568 3 PTEREVSIWR 0.060 1569 2 LPTEREVSIW 0.009 1570 8 VSIWRRSGPF 0.005 1571 10 IWRRSGPFAL 0.002 1572 7 EVSIWRRSGP 0.001 1573 6 REVSIWRRSG 0.000 1574 5 EREVSIWRRS 0.000 1575 158P3D2 v.5a A3-10mers SEQ. Pos 1234567890 Score ID NO. 44 AMGPGRGAIC 0.300 1576 12 SLPMTSLDPW 0.300 1577 2 LVLQVWDYTA 0.270 1578 1 VLVLQVWDYT 0.225 1579 40 CSWPAMGPGR 0.150 1580 16 TSLDPWSCSY 0.090 1581 4 LQVWDYTASL 0.081 1582 9 YTASLPMTSL 0.068 1583 38 ALCSWPAMGP 0.060 1584 14 PMTSLDPWSC 0.060 1585 3 VLQVWDYTAS 0.040 1586 29 CVGPGAPSSA 0.030 1587 17 SLDPWSCSYQ 0.030 1588 5 QVWDYTASLP 0.010 1589 25 YQTWCVGPGA 0.009 1590 46 GPGRGAICFA 0.009 1591 33 GAPSSALCSW 0.009 1592 45 MGPGRGAICF 0.006 1593 31 GPGAPSSALC 0.006 1594 19 DPWSCSYQTW 0.003 1595 15 MTSLDPWSCS 0.003 1596 21 WSCSYQTWCV 0.003 1597 48 GRGAICFAAA 0.002 1598 26 QTWCVGPGAP 0.002 1599 23 CSYQTWCVGP 0.002 1600 30 VGPGAPSSAL 0.001 1601 36 SSALCSWPAM 0.001 1602 28 WCVGPGAPSS 0.001 1603 37 SALCSWPAMG 0.001 1604 7 WDYTASLPMT 0.001 1605 49 RGAICFAAAA 0.001 1606 13 LPMTSLDPWS 0.001 1607 11 ASLPMTSLDP 0.000 1608 43 PAMGPGRGAI 0.000 1609 6 VWDYTASLPM 0.000 1610 18 LDPWSCSYQT 0.000 1611 42 WPAMGPGRGA 0.000 1612 35 PSSALCSWPA 0.000 1613 34 APSSALCSWP 0.000 1614 22 SCSYQTWCVG 0.000 1615 10 TASLPMTSLD 0.000 1616 47 PGRGAICFAA 0.000 1617 39 LCSWPAMGPG 0.000 1618 20 PWSCSYQTWC 0.000 1619 27 TWCVGPGAPS 0.000 1620 8 DYTASLPMTS 0.000 1621 24 SYQTWCVGPG 0.000 1622 32 PGAPSSALCS 0.000 1623 41 SWPAMGPGRG 0.000 1624

TABLE XI 158P3D2 A11, 9mers (variants 1, 2a, 3, 4 and 5a) 158P3D2 v.1 A11-9mers SEQ. Pos 123456789 Score ID NO. 320 QVIFRPLHK 6.000 1625 281 KTFVFFIWR 2.400 1626 59 WVKGLEHDK 2.000 1627 316 GQISQVIFR 1.080 1628 198 AQEAQAGKK 0.600 1629 53 DIYVKSWVK 0.480 1630 242 LLTVEEAEK 0.400 1631 21 RQPISYELR 0.360 1632 243 LTVEEAEKR 0.300 1633 201 AQAGKKKRK 0.300 1634 49 EMSSDIYVK 0.240 1635 227 GNVYILTGK 0.180 1636 123 VLQVWDYDR 0.160 1637 257 RKQPEPLEK 0.120 1638 90 RFDYLPTER 0.120 1639 282 TFVFFIWRR 0.120 1640 170 RCNLFRCRR 0.120 1641 285 FFIWRRYWR 0.120 1642 293 RTLVLLLLV 0.090 1643 300 LVLLTVFLL 0.090 1644 273 FNWFVNPLK 0.080 1645 181 GWWPVVKLK 0.060 1646 250 KRPVGKGRK 0.060 1647 109 FALEEAEFR 0.060 1648 247 EAEKRPVGK 0.060 1649 197 EAQEAQAGK 0.060 1650 235 KVEAEFELL 0.060 1651 142 ELQLPDMVR 0.048 1652 156 ELCSVQLAR 0.048 1653 145 LPDMVRGAR 0.040 1654 304 TVFLLLVFY 0.040 1655 82 NFNWRFVFR 0.040 1656 101 SVWRRSGPF 0.040 1657 228 NVYILTGKV 0.040 1658 162 LARNGAGPR 0.040 1659 295 LVLLLLVLL 0.030 1660 77 LTGEGNFNW 0.030 1661 199 QEAQAGKKK 0.030 1662 303 LTVFLLLVF 0.030 1663 38 VVLDDENPL 0.030 1664 30 VVIWNTEDV 0.030 1665 290 RYWRTLVLL 0.024 1666 276 FVNPLKTFV 0.020 1667 179 LRGWWPVVK 0.020 1668 11 PAPPPVDIK 0.020 1669 159 SVQLARNGA 0.020 1670 172 NLFRCRRLR 0.016 1671 204 GKKKRKQRR 0.012 1672 306 FLLLVFYTI 0.012 1673 301 VLLTVFLLL 0.012 1674 121 VLVLQVWDY 0.012 1675 96 TEREVSVWR 0.012 1676 178 RLRGWWPVV 0.012 1677 297 LLLLVLLTV 0.012 1678 294 TLVLLLLVL 0.012 1679 232 LTGKVEAEF 0.010 1680 222 FTDMGGNVY 0.010 1681 55 YVKSWVKGL 0.010 1682 46 LTGEMSSDI 0.010 1683 29 RVVIWNTED 0.009 1684 124 LQVWDYDRI 0.009 1685 270 KTSFNWFVN 0.009 1686 86 RFVFRFDYL 0.009 1687 319 SQVIFRPLH 0.009 1688 302 LLTVFLLLV 0.008 1689 87 FVFRFDYLP 0.008 1690 137 FLGSLELQL 0.008 1691 167 AGPRCNLFR 0.008 1692 31 VIWNTEDVV 0.008 1693 81 GNFNWRFVF 0.007 1694 48 GEMSSDIYV 0.007 1695 208 RKQRRRKGR 0.006 1696 206 KKRKQRRRK 0.006 1697 154 GPELCSVQL 0.006 1698 230 YILTGKVEA 0.006 1699 22 QPISYELRV 0.006 1700 299 LLVLLTVFL 0.006 1701 193 DVEREAQEA 0.006 1702 298 LLLVLLTVF 0.006 1703 265 KPSRPKTSF 0.006 1704 166 GAGPRCNLF 0.006 1705 200 EAQAGKKKR 0.006 1706 175 RCRRLRGWW 0.006 1707 268 RPKTSFNWF 0.006 1708 262 PLEKPSRPK 0.004 1709 25 SYELRVVIW 0.004 1710 2 WIDIFPQDV 0.004 1711 78 TGEGNFNWR 0.004 1712 188 LKEAEDVER 0.004 1713 309 LVFYTIPGQ 0.004 1714 118 QPAVLVLQV 0.004 1715 313 TIPGQISQV 0.004 1716 283 FVFFIWRRY 0.004 1717 310 VFYTIPGQI 0.004 1718 140 SLELQLPDM 0.004 1719 131 RISANDFLG 0.004 1720 79 GEGNFNWRF 0.004 1721 312 YTIPGQISQ 0.003 1722 278 NPLKTFVFF 0.003 1723 120 AVLVLQVWD 0.003 1724 158P3D2 v.2a A11-9mers SEQ. Pos 123456789 Score ID NO. 109 GIPQNRPIK 1.200 1725 153 DTKERYIPK 0.600 1726 118 LLVRVYVVK 0.600 1727 26 EVKGTVSPK 0.600 1728 130 LAPADPNGK 0.200 1729 57 WLNVFPLYR 0.160 1730 37 VATLKIYNR 0.080 1731 100 LFSEPQISR 0.080 1732 201 ETHIDLENR 0.060 1733 143 VVVSAGRER 0.060 1734 117 KLLVRVYVV 0.036 1735 98 AVLFSEPQI 0.030 1736 123 YVVKATNLA 0.030 1737 29 GTVSPKKAV 0.022 1738 27 VKGTVSPKK 0.020 1739 30 TVSPKKAVA 0.020 1740 184 TVAVFEHDL 0.020 1741 147 AGRERQDTK 0.020 1742 82 LVGKFKGSF 0.020 1743 20 QDQGEAEVK 0.020 1744 119 LVRVYVVKA 0.020 1745 149 RERQDTKER 0.018 1746 141 PYVVVSAGR 0.012 1747 85 KFKGSFLIY 0.012 1748 219 GLASQYEVW 0.012 1749 121 RVYVVKATN 0.012 1750 9 GVNLISMVG 0.012 1751 79 SGHLVGKFK 0.010 1752 114 RPIKLLVRV 0.009 1753 161 KQLNPIFGE 0.008 1754 186 AVFEHDLVG 0.008 1755 113 NRPIKLLVR 0.008 1756 91 LIYPESEAV 0.008 1757 198 LIGETHIDL 0.008 1758 228 VQQGPQEPF 0.006 1759 19 IQDQGEAEV 0.006 1760 90 FLIYPESEA 0.006 1761 11 NLISMVGEI 0.006 1762 77 EGSGHLVGK 0.006 1763 122 VYVVKATNL 0.006 1764 41 KIYNRSLEE 0.005 1765 35 KAVATLKIY 0.005 1766 124 VVKATNLAP 0.004 1767 46 SLEEEFNHF 0.004 1768 177 LPAETELTV 0.004 1769 158 YIPKQLNPI 0.004 1770 106 ISRGIPQNR 0.004 1771 192 LVGSDDLIG 0.004 1772 36 AVATLKIYN 0.004 1773 110 IPQNRPIKL 0.004 1774 92 IYPESEAVL 0.004 1775 162 QLNPIFGEI 0.004 1776 84 GKFKGSFLI 0.004 1777 157 RYIPKQLNP 0.004 1778 169 EILELSISL 0.004 1779 182 ELTVAVFEH 0.004 1780 185 VAVFEHDLV 0.003 1781 180 ETELTVAVF 0.003 1782 142 YVVVSAGRE 0.003 1783 45 RSLEEEFNH 0.003 1784 17 GEIQDQGEA 0.003 1785 207 ENRFYSHHR 0.002 1786 205 DLENRFYSH 0.002 1787 33 PKKAVATLK 0.002 1788 144 VVSAGRERQ 0.002 1789 159 IPKQLNPIF 0.002 1790 53 HFEDWLNVF 0.002 1791 32 SPKKAVATL 0.002 1792 227 WVQQGPQEP 0.002 1793 131 APADPNGKA 0.002 1794 220 LASQYEVWV 0.002 1795 15 MVGEIQDQG 0.002 1796 93 YPESEAVLF 0.002 1797 151 RQDTKERYI 0.002 1798 69 GQDGGGEEE 0.002 1799 66 GQGGQDGGG 0.002 1800 23 GEAEVKGTV 0.002 1801 74 GEEEGSGHL 0.002 1802 171 LELSISLPA 0.002 1803 191 DLVGSDDLI 0.002 1804 165 PIFGEILEL 0.002 1805 183 LTVAVFEHD 0.002 1806 38 ATLKIYNRS 0.002 1807 225 EVWVQQGPQ 0.001 1808 34 KKAVATLKI 0.001 1809 222 SQYEVWVQQ 0.001 1810 127 ATNLAPADP 0.001 1811 155 KERYIPKQL 0.001 1812 99 VLFSEPQIS 0.001 1813 112 QNRPIKLLV 0.001 1814 52 NHFEDWLNV 0.001 1815 126 KATNLAPAD 0.001 1816 138 KADPYVVVS 0.001 1817 78 GSGHLVGKF 0.001 1818 14 SMVGEIQDQ 0.001 1819 218 CGLASQYEV 0.001 1820 73 GGEEEGSGH 0.001 1821 215 RANCGLASQ 0.001 1822 164 NPIFGEILE 0.001 1823 5 GDSDGVNLI 0.001 1824 158P3D2 v.3 A11-9mers SEQ. Pos 123456789 Score ID NO. 1 PTEREVSVR 0.020 1825 7 SVRRRSGPF 0.020 1826 2 TEREVSVRR 0.012 1827 9 RRRSGPFAL 0.002 1828 8 VRRRSGPFA 0.000 1829 3 EREVSVRRR 0.000 1830 5 EVSVRRRSG 0.000 1831 6 VSVRRRSGP 0.000 1832 4 REVSVRRRS 0.000 1833 158P3D2 v.4 A11-9mers SEQ. Pos 123456789 Score ID NO. 3 TEREVSIWR 0.024 1834 8 SIWRRSGPF 0.008 1835 4 EREVSIWRR 0.002 1836 1 LPTEREVSI 0.002 1837 2 PTEREVSIW 0.001 1838 9 IWRRSGPFA 0.000 1839 6 EVSIWRRSG 0.000 1840 7 VSIWRRSGP 0.000 1841 5 REVSIWRRS 0.000 1842 158P3D2 v.5a A11-9mers SEQ. Pos 123456789 Score ID NO. 4 QVWDYTASL 0.040 1843 25 QTWCVGPGA 0.020 1844 45 GPGRGAICF 0.012 1845 49 GAICFAAAA 0.009 1846 2 VLQVWDYTA 0.008 1847 30 GPGAPSSAL 0.006 1848 16 SLDPWSCSY 0.004 1849 21 SCSYQTWCV 0.004 1850 43 AMGPGRGAI 0.004 1851 40 SWPAMGPGR 0.004 1852 12 LPMTSLDPW 0.004 1853 1 LVLQVWDYT 0.003 1854 36 SALCSWPAM 0.003 1855 14 MTSLDPWSC 0.002 1856 9 TASLPMTSL 0.002 1857 33 APSSALCSW 0.002 1858 28 CVGPGAPSS 0.002 1859 8 YTASLPMTS 0.002 1860 47 GRGAICFAA 0.002 1861 32 GAPSSALCS 0.001 1862 3 LQVWDYTAS 0.001 1863 11 SLPMTSLDP 0.001 1864 6 WDYTASLPM 0.001 1865 24 YQTWCVGPG 0.001 1866 48 RGAICFAAA 0.001 1867 37 ALCSWPAMG 0.000 1868 38 LCSWPAMGP 0.000 1869 23 SYQTWCVGP 0.000 1870 35 SSALCSWPA 0.000 1871 27 WCVGPGAPS 0.000 1872 18 DPWSCSYQT 0.000 1873 41 WPAMGPGRG 0.000 1874 29 VGPGAPSSA 0.000 1875 7 DYTASLPMT 0.000 1876 22 CSYQTWCVG 0.000 1877 13 PMTSLDPWS 0.000 1878 39 CSWPAMGPG 0.000 1879 42 PAMGPGRGA 0.000 1880 15 TSLDPWSCS 0.000 1881 10 ASLPMTSLD 0.000 1882 26 TWCVGPGAP 0.000 1883 20 WSCSYQTWC 0.000 1884 5 VWDYTASLP 0.000 1885 46 PGRGAICFA 0.000 1886 19 PWSCSYQTW 0.000 1887 17 LDPWSCSYQ 0.000 1888 44 MGPGRGAIC 0.000 1889 34 PSSALCSWP 0.000 1890 31 PGAPSSALC 0.000 1891

TABLE XII 158P3D2 A11, 10mers (variants 1, 2a, 3, 4 and 5a) 158P3D2 v.1 A11- 10mers SEQ. ID Pos 1234567890 Score NO. 281 KTFVFFIWRR 2.400 1892 319 SQVIFRPLHK 1.800 1893 122 LVLQVWDYDR 1.200 1894 178 RLRGWWPVVK 1.200 1895 48 GEMSSDIYVK 0.720 1896 198 AQEAQAGKKK 0.300 1897 166 GAGPRCNLFR 0.240 1898 187 KLKEAEDVER 0.240 1899 272 SFNWFVNPLK 0.200 1900 10 VPAPPPVDIK 0.200 1901 77 LTGEGNFNWR 0.200 1902 241 ELLTVEEAEK 0.180 1903 196 REAQEAQAGK 0.180 1904 284 VFFIWRRYWR 0.160 1905 256 GRKQPEPLEK 0.120 1906 29 RVVIWNTEDV 0.090 1907 293 RTLVLLLLVL 0.090 1908 125 QVWDYDRISA 0.080 1909 144 QLPDMVRGAR 0.080 1910 161 QLARNGAGPR 0.080 1911 242 LLTVEEAEKR 0.080 1912 226 GGNVYILTGK 0.060 1913 180 RGWWPVVKLK 0.060 1914 52 SDIYVKSWVK 0.060 1915 120 AVLVLQVWDY 0.060 1916 300 LVLLTVFLLL 0.060 1917 197 EAQEAQAGKK 0.060 1918 276 FVNPLKTFVF 0.060 1919 81 GNFNWRFVFR 0.048 1920 290 RYWRTLVLLL 0.048 1921 101 SVWRRSGPFA 0.040 1922 259 QPEPLEKPSR 0.040 1923 309 LVFYTIPGQI 0.040 1924 21 RQPISYELRV 0.036 1925 141 LELQLPDMVR 0.036 1926 117 RQPAVLVLQV 0.036 1927 58 SWVKGLEHDK 0.030 1928 200 EAQAGKKKRK 0.030 1929 30 VVIWNTEDVV 0.030 1930 96 TEREVSVWRR 0.024 1931 12 APPPVDIKPR 0.020 1932 148 MVRGARGPEL 0.020 1933 202 QAGKKKRKQR 0.020 1934 185 VVKLKEAEDV 0.020 1935 95 PTEREVSVWR 0.020 1936 299 LLVLLTVFLL 0.018 1937 246 EEAEKRPVGK 0.018 1938 303 LTVFLLLVFY 0.015 1939 312 YTIPGQISQV 0.015 1940 168 GPRCNLFRCR 0.012 1941 235 KVEAEFELLT 0.012 1942 19 KPRQPISYEL 0.012 1943 304 TVFLLLVFYT 0.012 1944 296 VLLLLVLLTV 0.012 1945 107 GPFALEEAEF 0.012 1946 76 SLTGEGNFNW 0.012 1947 301 VLLTVFLLLV 0.012 1948 268 RPKTSFNWFV 0.012 1949 222 FTDMGGNVYI 0.010 1950 46 LTGEMSSDIY 0.010 1951 37 DVVLDDENPL 0.009 1952 261 EPLEKPSRPK 0.009 1953 278 NPLKTFVFFI 0.009 1954 316 GQISQVIFRP 0.008 1955 280 LKTFVFFIWR 0.008 1956 87 FVFRFDYLPT 0.008 1957 302 LLTVFLLLVF 0.008 1958 89 FRFDYLPTER 0.008 1959 31 VIWNTEDVVL 0.008 1960 207 KRKQRRRKGR 0.006 1961 205 KKKRKQRRRK 0.006 1962 216 RPEDLEFTDM 0.006 1963 249 EKRPVGKGRK 0.006 1964 294 TLVLLLLVLL 0.006 1965 305 VFLLLVFYTI 0.006 1966 82 NFNWRFVFRF 0.006 1967 297 LLLLVLLTVF 0.006 1968 199 QEAQAGKKKR 0.006 1969 295 LVLLLLVLLT 0.006 1970 154 GPELCSVQLA 0.006 1971 151 GARGPELCSV 0.006 1972 9 DVPAPPPVDI 0.006 1973 298 LLLVLLTVFL 0.006 1974 248 AEKRPVGKGR 0.006 1975 229 VYILTGKVEA 0.006 1976 67 KQETDVHFNS 0.005 1977 123 VLQVWDYDRI 0.004 1978 93 YLPTEREVSV 0.004 1979 283 FVFFIWRRYW 0.004 1980 203 AGKKKRKQRR 0.004 1981 231 ILTGKVEAEF 0.004 1982 108 PFALEEAEFR 0.004 1983 140 SLELQLPDMV 0.004 1984 313 TIPGQISQVI 0.004 1985 155 PELCSVQLAR 0.004 1986 38 VVLDDENPLT 0.003 1987 275 WFVNPLKTFV 0.003 1988 270 KTSFNWFVNP 0.003 1989 54 IYVKSWVKGL 0.003 1990 131 RISANDFLGS 0.002 1991 158P3D2 v.2a A11- 10-mers SEQ. ID Pos 1234567890 Score NO. 117 KLLVRVYVVK 1.800 1992 36 AVATLKIYNR 0.800 1993 19 IQDQGEAEVK 0.600 1994 26 EVKGTVSPKK 0.600 1995 129 NLAPADPNGK 0.400 1996 99 VLFSEPQISR 0.320 1997 32 SPKKAVATLK 0.200 1998 146 SAGRERQDTK 0.200 1999 121 RVYVVKATNL 0.120 2000 108 RGIPQNRPIK 0.090 2001 25 AEVKGTVSPK 0.090 2002 105 QISRGIPQNR 0.080 2003 142 YVVVSAGRER 0.060 2004 29 GTVSPKKAVA 0.045 2005 152 QDTKERYIPK 0.040 2006 200 GETHIDLENR 0.036 2007 78 GSGHLVGKFK 0.030 2008 161 KQLNPIFGEI 0.027 2009 140 DPYVVVSAGR 0.024 2010 109 GIPQNRPIKL 0.024 2011 227 WVQQGPQEPF 0.020 2012 82 LVGKFKGSFL 0.020 2013 184 TVAVFEHDLV 0.020 2014 124 VVKATNLAPA 0.020 2015 76 EEGSGHLVGK 0.018 2016 157 RYIPKQLNPI 0.018 2017 112 QNRPIKLLVR 0.016 2018 183 LTVAVFEHDL 0.015 2019 219 GLASQYEVWV 0.012 2020 206 LENRFYSHHR 0.012 2021 170 ILELSISLPA 0.008 2022 176 SLPAETELTV 0.008 2023 91 LIYPESEAVL 0.008 2024 148 GRERQDTKER 0.006 2025 164 NPIFGEILEL 0.006 2026 122 VYVVKATNLA 0.006 2027 9 GVNLISMVGE 0.006 2028 118 LLVRVYVVKA 0.006 2029 81 HLVGKFKGSF 0.006 2030 138 KADPYVVVSA 0.006 2031 215 RANCGLASQY 0.006 2032 123 YVVKATNLAP 0.006 2033 90 FLIYPESEAV 0.006 2034 168 GEILELSISL 0.005 2035 59 NVFPLYRGQG 0.004 2036 186 AVFEHDLVGS 0.004 2037 42 IYNRSLEEEF 0.004 2038 217 NCGLASQYEV 0.004 2039 166 IFGEILELSI 0.004 2040 192 LVGSDDLIGE 0.004 2041 174 SISLPAETEL 0.004 2042 158 YIPKQLNPIF 0.004 2043 162 QLNPIFGEIL 0.004 2044 92 IYPESEAVLF 0.004 2045 151 RQDTKERYIP 0.004 2046 197 DLIGETHIDL 0.004 2047 56 DWLNVFPLYR 0.004 2048 143 VVVSAGRERQ 0.003 2049 201 ETHIDLENRF 0.003 2050 89 SFLIYPESEA 0.003 2051 98 AVLFSEPQIS 0.003 2052 181 TELTVAVFEH 0.003 2053 41 KIYNRSLEEE 0.002 2054 84 GKFKGSFLIY 0.002 2055 130 LAPADPNGKA 0.002 2056 30 TVSPKKAVAT 0.002 2057 15 MVGEIQDQGE 0.002 2058 177 LPAETELTVA 0.002 2059 66 GQGGQDGGGE 0.002 2060 35 KAVATLKIYN 0.002 2061 69 GQDGGGEEEG 0.002 2062 54 FEDWLNVFPL 0.002 2063 74 GEEEGSGHLV 0.002 2064 149 RERQDTKERY 0.002 2065 38 ATLKIYNRSL 0.002 2066 203 HIDLENRFYS 0.001 2067 85 KFKGSFLIYP 0.001 2068 209 RFYSHHRANC 0.001 2069 222 SQYEVWVQQG 0.001 2070 111 PQNRPIKLLV 0.001 2071 225 EVWVQQGPQE 0.001 2072 18 EIQDQGEAEV 0.001 2073 205 DLENRFYSHH 0.001 2074 110 IPQNRPIKLL 0.001 2075 119 LVRVYVVKAT 0.001 2076 127 ATNLAPADPN 0.001 2077 45 RSLEEEFNHF 0.001 2078 114 RPIKLLVRVY 0.001 2079 97 EAVLFSEPQI 0.001 2080 57 WLNVFPLYRG 0.001 2081 51 FNHFEDWLNV 0.001 2082 12 LISMVGEIQD 0.001 2083 14 SMVGEIQDQG 0.001 2084 116 IKLLVRVYVV 0.001 2085 73 GGEEEGSGHL 0.001 2086 44 NRSLEEEFNH 0.001 2087 10 VNLISMVGEI 0.001 2088 126 KATNLAPADP 0.001 2089 194 GSDDLIGETH 0.001 2090 83 VGKFKGSFLI 0.001 2091 158P3D2 v.3 A11- 10mers SEQ. ID Pos 1234567890 Score NO. 1 LPTEREVSVR 0.040 2092 8 SVRRRSGPFA 0.020 2093 2 PTEREVSVRR 0.020 2094 3 TEREVSVRRR 0.001 2095 6 EVSVRRRSGP 0.001 2096 9 VRRRSGPFAL 0.001 2097 7 VSVRRRSGPF 0.000 2098 10 RRRSGPFALE 0.000 2099 5 REVSVRRRSG 0.000 2100 4 EREVSVRRRS 0.000 2101 158P3D2 v.4 A11-10mers SEQ. ID Pos 1234567890 Score NO. 3 PTEREVSIWR 0.040 2102 4 TEREVSIWRR 0.024 2103 9 SIWRRSGPFA 0.008 2104 1 YLPTEREVSI 0.004 2105 2 LPTEREVSIW 0.002 2106 7 EVSIWRRSGP 0.001 2107 10 IWRRSGPFAL 0.001 2108 8 VSIWRRSGPF 0.000 2109 6 REVSIWRRSG 0.000 2110 5 EREVSIWRRS 0.000 2111 158P3D2 v.5a A11- 10mers SEQ. ID Pos 1234567890 Score NO. 2 LVLQVWDYTA 0.060 2112 29 CVGPGAPSSA 0.020 2113 9 YTASLPMTSL 0.010 2114 4 LQVWDYTASL 0.009 2115 40 CSWPAMGPGR 0.008 2116 46 GPGRGAICFA 0.006 2117 25 YQTWCVGPGA 0.006 2118 33 GAPSSALCSW 0.006 2119 5 QVWDYTASLP 0.004 2120 12 SLPMTSLDPW 0.004 2121 26 QTWCVGPGAP 0.002 2122 19 DPWSCSYQTW 0.001 2123 15 MTSLDPWSCS 0.001 2124 38 ALCSWPAMGP 0.001 2125 1 VLVLQVWDYT 0.001 2126 48 GRGAICFAAA 0.001 2127 49 RGAICFAAAA 0.001 2128 31 GPGAPSSALC 0.001 2129 45 MGPGRGAICF 0.000 2130 6 VWDYTASLPM 0.000 2131 21 WSCSYQTWCV 0.000 2132 43 PAMGPGRGAI 0.000 2133 44 AMGPGRGAIC 0.000 2134 3 VLQVWDYTAS 0.000 2135 13 LPMTSLDPWS 0.000 2136 24 SYQTWCVGPG 0.000 2137 17 SLDPWSCSYQ 0.000 2138 16 TSLDPWSCSY 0.000 2139 37 SALCSWPAMG 0.000 2140 28 WCVGPGAPSS 0.000 2141 8 DYTASLPMTS 0.000 2142 34 APSSALCSWP 0.000 2143 22 SCSYQTWCVG 0.000 2144 10 TASLPMTSLD 0.000 2145 30 VGPGAPSSAL 0.000 2146 36 SSALCSWPAM 0.000 2147 42 WPAMGPGRGA 0.000 2148 39 LCSWPAMGPG 0.000 2149 14 PMTSLDPWSC 0.000 2150 11 ASLPMTSLDP 0.000 2151 47 PGRGAICFAA 0.000 2152 23 CSYQTWCVGP 0.000 2153 7 WDYTASLPMT 0.000 2154 18 LDPWSCSYQT 0.000 2155 35 PSSALCSWPA 0.000 2156 41 SWPAMGPGRG 0.000 2157 27 TWCVGPGAPS 0.000 2158 32 PGAPSSALCS 0.000 2159 20 PWSCSYQTWC 0.000 2160

TABLE XIII 158P3D2 A24, 9mers (variants 1, 2a, 3, 4 and 5a) 158P3D2 v.1 A24- 9mers SEQ. Pos 123456789 Score ID NO. 268 RPKTSFNWF 120.000 2161 265 KPSRPKTSF 40.000 2162 278 NPLKTFVFF 20.000 2163 214 KGRPEDLEF 9.000 2164 314 IPGQISQVI 8.000 2165 94 LPTEREVSV 8.000 2166 10 VPAPPPVDI 8.000 2167 154 GPELCSVQL 6.000 2168 168 GPRCNLFRC 6.000 2169 255 KGRKQPEPL 6.000 2170 133 SANDFLGSL 6.000 2171 318 ISQVIFRPL 5.000 2172 75 NSLTGEGNF 5.000 2173 118 QPAVLVLQV 4.000 2174 24 ISYELRVVI 4.000 2175 22 QPISYELRV 4.000 2176 38 VVLDDENPL 3.000 2177 55 YVKSWVKGL 3.000 2178 175 RCRRLRGWW 3.000 2179 166 GAGPRCNLF 3.000 2180 180 RGWWPVVKL 2.000 2181 183 WPVVKLKEA 2.000 2182 283 FVFFIWRRY 2.000 2183 304 TVFLLLVFY 2.000 2184 121 VLVLQVWDY 2.000 2185 44 NPLTGEMSS 2.000 2186 19 KPRQPISYE 1.200 2187 178 RLRGWWPVV 1.200 2188 299 LLVLLTVFL 1.000 2189 165 NGAGPRCNL 1.000 2190 224 DMGGNVYIL 1.000 2191 277 VNPLKTFVF 1.000 2192 298 LLLVLLTVF 1.000 2193 294 TLVLLLLVL 1.000 2194 137 FLGSLELQL 1.000 2195 171 CNLFRCRRL 1.000 2196 101 SVWRRSGPF 1.000 2197 81 GNFNWRFVF 1.000 2198 300 LVLLTVFLL 1.000 2199 50 MSSDIYVKS 1.000 2200 83 FNWRFVFRF 1.000 2201 232 LTGKVEAEF 1.000 2202 303 LTVFLLLVF 1.000 2203 301 VLLTVFLLL 1.000 2204 295 LVLLLLVLL 1.000 2205 77 LTGEGNFNW 1.000 2206 235 KVEAEFELL 0.900 2207 151 GARGPELCS 0.900 2208 46 LTGEMSSDI 0.800 2209 51 SSDIYVKSW 0.750 2210 132 ISANDFLGS 0.750 2211 222 FTDMGGNVY 0.600 2212 47 TGEMSSDIY 0.600 2213 259 QPEPLEKPS 0.600 2214 140 SLELQLPDM 0.600 2215 212 RRKGRPEDL 0.600 2216 124 LQVWDYDRI 0.600 2217 293 RTLVLLLLV 0.400 2218 306 FLLLVFYTI 0.400 2219 251 RPVGKGRKQ 0.400 2220 6 FPQDVPAPP 0.400 2221 261 EPLEKPSRP 0.400 2222 129 YDRISANDF 0.300 2223 291 YWRTLVLLL 0.300 2224 17 DIKPRQPIS 0.300 2225 27 ELRVVIWNT 0.300 2226 287 IWRRYWRTL 0.300 2227 69 ETDVHFNSL 0.300 2228 103 WRRSGPFAL 0.300 2229 237 EAEFELLTV 0.270 2230 216 RPEDLEFTD 0.240 2231 164 RNGAGPRCN 0.200 2232 234 GKVEAEFEL 0.200 2233 30 VVIWNTEDV 0.200 2234 313 TIPGQISQV 0.200 2235 18 IKPRQPISY 0.200 2236 150 RGARGPELC 0.200 2237 297 LLLLVLLTV 0.200 2238 42 DENPLTGEM 0.200 2239 107 GPFALEEAE 0.200 2240 290 RYWRTLVLL 0.200 2241 302 LLTVFLLLV 0.200 2242 12 APPPVDIKP 0.200 2243 31 VIWNTEDVV 0.200 2244 276 FVNPLKTFV 0.200 2245 228 NVYILTGKV 0.200 2246 125 QVWDYDRIS 0.200 2247 86 RFVFRFDYL 0.200 2248 144 QLPDMVRGA 0.200 2249 66 DKQETDVHF 0.200 2250 80 EGNFNWRFV 0.200 2251 85 WRFVFRFDY 0.200 2252 289 RRYWRTLVL 0.200 2253 270 KTSFNWFVN 0.200 2254 113 EAEFRQPAV 0.180 2255 190 EAEDVEREA 0.180 2256 76 SLTGEGNFN 0.150 2257 266 PSRPKTSFN 0.150 2258 32 IWNTEDVVL 0.150 2259 119 PAVLVLQVW 0.150 2260 158P3D2 v.2a A24- 9mers SEQ. Pos 123456789 Score ID NO. 92 IYPESEAVL 360.000 2261 122 VYVVKATNL 300.000 2262 50 EFNHFEDWL 30.000 2263 53 HFEDWLNVF 21.600 2264 169 EILELSISL 8.640 2265 175 ISLPAETEL 7.920 2266 110 IPQNRPIKL 6.600 2267 163 LNPIFGEIL 6.000 2268 46 SLEEEFNHF 5.184 2269 210 FYSHHRANC 5.000 2270 198 LIGETHIDL 4.800 2271 83 VGKFKGSFL 4.000 2272 32 SPKKAVATL 4.000 2273 39 TLKIYNRSL 4.000 2274 184 TVAVFEHDL 4.000 2275 108 RGIPQNRPI 3.600 2276 162 QLNPIFGEI 3.326 2277 228 VQQGPQEPF 3.000 2278 180 ETELTVAVF 3.000 2279 93 YPESEAVLF 3.000 2280 43 YNRSLEEEF 2.640 2281 78 GSGHLVGKF 2.640 2282 159 IPKQLNPIF 2.400 2283 82 LVGKFKGSF 2.000 2284 151 RQDTKERYI 2.000 2285 167 FGEILELSI 1.800 2286 157 RYIPKQLNP 1.800 2287 158 YIPKQLNPI 1.800 2288 11 NLISMVGEI 1.650 2289 191 DLVGSDDLI 1.500 2290 98 AVLFSEPQI 1.500 2291 85 KFKGSFLIY 1.200 2292 155 KERYIPKQL 1.120 2293 209 RFYSHHRAN 1.000 2294 223 QYEVWVQQG 0.900 2295 166 IFGEILELS 0.840 2296 42 IYNRSLEEE 0.825 2297 187 VFEHDLVGS 0.750 2298 74 GEEEGSGHL 0.720 2299 190 HDLVGSDDL 0.600 2300 111 PQNRPIKLL 0.600 2301 202 THIDLENRF 0.518 2302 63 LYRGQGGQD 0.500 2303 165 PIFGEILEL 0.440 2304 4 PGDSDGVNL 0.400 2305 55 EDWLNVFPL 0.400 2306 212 SHHRANCGL 0.400 2307 114 RPIKLLVRV 0.360 2308 117 KLLVRVYVV 0.300 2309 35 KAVATLKIY 0.300 2310 121 RVYVVKATN 0.280 2311 38 ATLKIYNRS 0.252 2312 56 DWLNVFPLY 0.252 2313 138 KADPYVVVS 0.240 2314 34 KKAVATLKI 0.220 2315 28 KGTVSPKKA 0.220 2316 102 SEPQISRGI 0.210 2317 173 LSISLPAET 0.198 2318 81 HLVGKFKGS 0.180 2319 123 YVVKATNLA 0.180 2320 8 DGVNLISMV 0.180 2321 112 QNRPIKLLV 0.168 2322 218 CGLASQYEV 0.165 2323 90 FLIYPESEA 0.165 2324 194 GSDDLIGET 0.158 2325 88 GSFLIYPES 0.154 2326 31 VSPKKAVAT 0.150 2327 185 VAVFEHDLV 0.150 2328 24 EAEVKGTVS 0.150 2329 29 GTVSPKKAV 0.150 2330 196 DDLIGETHI 0.150 2331 134 DPNGKADPY 0.150 2332 176 SLPAETELT 0.150 2333 128 TNLAPADPN 0.150 2334 22 QGEAEVKGT 0.150 2335 5 GDSDGVNLI 0.144 2336 6 DSDGVNLIS 0.140 2337 131 APADPNGKA 0.132 2338 36 AVATLKIYN 0.120 2339 99 VLFSEPQIS 0.120 2340 30 TVSPKKAVA 0.120 2341 146 SAGRERQDT 0.120 2342 91 LIYPESEAV 0.120 2343 177 LPAETELTV 0.120 2344 3 DPGDSDGVN 0.120 2345 216 ANCGLASQY 0.120 2346 119 LVRVYVVKA 0.110 2347 19 IQDQGEAEV 0.110 2348 141 PYVVVSAGR 0.105 2349 220 LASQYEVWV 0.100 2350 136 NGKADPYVV 0.100 2351 51 FNHFEDWLN 0.100 2352 84 GKFKGSFLI 0.100 2353 219 GLASQYEVW 0.100 2354 71 DGGGEEEGS 0.100 2355 105 QISRGIPQN 0.100 2356 203 HIDLENRFY 0.100 2357 89 SFLIYPESE 0.075 2358 60 VFPLYRGQG 0.075 2359 100 LFSEPQISR 0.060 2360 158P3D2 v.3 A24- 9mers SEQ. Pos 123456789 Score ID NO. 7 SVRRRSGPF 2.000 2361 9 RRRSGPFAL 0.800 2362 4 REVSVRRRS 0.042 2363 6 VSVRRRSGP 0.015 2364 8 VRRRSGPFA 0.010 2365 5 EVSVRRRSG 0.010 2366 2 TEREVSVRR 0.002 2367 3 EREVSVRRR 0.002 2368 1 PTEREVSVR 0.002 2369 158P3D2 v.4 A24- 9mers SEQ. Pos 123456789 Score ID NO. 8 SIWRRSGPF 2.000 2370 1 LPTEREVSI 1.200 2371 9 IWRRSGPFA 0.100 2372 5 REVSIWRRS 0.042 2373 7 VSIWRRSGP 0.015 2374 2 PTEREVSIW 0.015 2375 6 EVSIWRRSG 0.010 2376 3 TEREVSIWR 0.002 2377 4 EREVSIWRR 0.002 2378 158P3D2 v.5a A24- 9mers SEQ. Pos 123456789 Score ID NO. 7 DYTASLPMT 5.000 2379 4 QVWDYTASL 4.800 2380 30 GPGAPSSAL 4.000 2381 9 TASLPMTSL 4.000 2382 45 GPGRGAICF 2.000 2383 43 AMGPGRGAI 1.200 2384 23 SYQTWCVGP 0.750 2385 36 SALCSWPAM 0.750 2386 48 RGAICFAAA 0.240 2387 1 LVLQVWDYT 0.210 2388 15 TSLDPWSCS 0.180 2389 29 VGPGAPSSA 0.150 2390 27 WCVGPGAPS 0.150 2391 3 LQVWDYTAS 0.150 2392 12 LPMTSLDPW 0.150 2393 32 GAPSSALCS 0.150 2394 49 GAICFAAAA 0.150 2395 44 MGPGRGAIC 0.150 2396 2 VLQVWDYTA 0.150 2397 25 QTWCVGPGA 0.140 2398 8 YTASLPMTS 0.120 2399 16 SLDPWSCSY 0.120 2400 28 CVGPGAPSS 0.120 2401 14 MTSLDPWSC 0.100 2402 33 APSSALCSW 0.100 2403 35 SSALCSWPA 0.100 2404 21 SCSYQTWCV 0.100 2405 18 DPWSCSYQT 0.100 2406 20 WSCSYQTWC 0.100 2407 6 WDYTASLPM 0.050 2408 10 ASLPMTSLD 0.018 2409 40 SWPAMGPGR 0.015 2410 11 SLPMTSLDP 0.015 2411 42 PAMGPGRGA 0.015 2412 47 GRGAICFAA 0.014 2413 19 PWSCSYQTW 0.012 2414 13 PMTSLDPWS 0.012 2415 31 PGAPSSALC 0.012 2416 39 CSWPAMGPG 0.012 2417 24 YQTWCVGPG 0.010 2418 41 WPAMGPGRG 0.010 2419 5 VWDYTASLP 0.010 2420 22 CSYQTWCVG 0.010 2421 46 PGRGAICFA 0.010 2422 37 ALCSWPAMG 0.010 2423 26 TWCVGPGAP 0.010 2424 38 LCSWPAMGP 0.010 2425 17 LDPWSCSYQ 0.002 2426 34 PSSALCSWP 0.001 2427

TABLE XIV 158P3D2 A24, 10mers (variants 1, 2a, 3, 4 and 5a) 158P3D2 v.1 A24- 10mers SEQ. ID Pos 1234567890 Score NO. 290 RYWRTLVLLL 480.000 2428 54 IYVKSWVKGL 300.000 2429 128 DYDRISANDF 120.000 2430 136 DFLGSLELQL 36.000 2431 115 EFRQPAVLVL 20.000 2432 82 NFNWRFVFRF 15.000 2433 153 RGPELCSVQL 14.400 2434 293 RTLVLLLLVL 14.400 2435 305 VFLLLVFYTI 12.600 2436 19 KPRQPISYEL 12.320 2437 170 RCNLFRCRRL 12.000 2438 25 SYELRVVIWN 10.500 2439 300 LVLLTVFLLL 10.080 2440 92 DYLPTEREVS 9.000 2441 229 VYILTGKVEA 8.250 2442 164 RNGAGPRCNL 8.000 2443 37 DVVLDDENPL 7.200 2444 294 TLVLLLLVLL 7.200 2445 298 LLLVLLTVFL 7.200 2446 317 QISQVIFRPL 6.720 2447 299 LLVLLTVFLL 6.000 2448 113 EAEFRQPAVL 6.000 2449 291 YWRTLVLLLL 5.600 2450 271 TSFNWFVNPL 4.800 2451 134 ANDFLGSLEL 4.400 2452 233 TGKVEAEFEL 4.400 2453 148 MVRGARGPEL 4.400 2454 31 VIWNTEDVVL 4.000 2455 286 FIWRRYWRTL 4.000 2456 132 ISANDFLGSL 4.000 2457 102 VWRRSGPFAL 4.000 2458 277 VNPLKTFVFF 3.600 2459 276 FVNPLKTFVF 3.600 2460 297 LLLLVLLTVF 3.600 2461 231 ILTGKVEAEF 3.080 2462 80 EGNFNWRFVF 3.000 2463 78 TGEGNFNWRF 3.000 2464 100 VSVWRRSGPF 3.000 2465 313 TIPGQISQVI 2.520 2466 302 LLTVFLLLVF 2.400 2467 165 NGAGPRCNLF 2.400 2468 107 GPFALEEAEF 2.200 2469 216 RPEDLEFTDM 2.160 2470 314 IPGQISQVIF 2.000 2471 74 FNSLTGEGNF 2.000 2472 274 NWFVNPLKTF 2.000 2473 9 DVPAPPPVDI 1.500 2474 278 NPLKTFVFFI 1.500 2475 123 VLQVWDYDRI 1.500 2476 309 LVFYTIPGQI 1.400 2477 282 TFVFFIWRRY 1.050 2478 222 FTDMGGNVYI 1.000 2479 275 WFVNPLKTFV 0.900 2480 139 GSLELQLPDM 0.900 2481 234 GKVEAEFELL 0.864 2482 239 EFELLTVEEA 0.825 2483 211 RRRKGRPEDL 0.800 2484 289 RRYWRTLVLL 0.800 2485 285 FFIWRRYWRT 0.750 2486 73 HFNSLTGEGN 0.750 2487 221 EFTDMGGNVY 0.720 2488 68 QETDVHFNSL 0.691 2489 223 TDMGGNVYIL 0.600 2490 310 VFYTIPGQIS 0.600 2491 311 FYTIPGQISQ 0.500 2492 173 LFRCRRLRGW 0.500 2493 85 WRFVFRFDYL 0.480 2494 61 KGLEHDKQET 0.475 2495 213 RKGRPEDLEF 0.440 2496 179 LRGWWPVVKL 0.440 2497 267 SRPKTSFNWF 0.432 2498 258 KQPEPLEKPS 0.432 2499 67 KQETDVHFNS 0.420 2500 129 YDRISANDFL 0.400 2501 254 GKGRKQPEPL 0.400 2502 288 WRRYWRTLVL 0.400 2503 117 RQPAVLVLQV 0.360 2504 29 RVVIWNTEDV 0.300 2505 235 KVEAEFELLT 0.300 2506 264 EKPSRPKTSF 0.300 2507 21 RQPISYELRV 0.300 2508 105 RSGPFALEEA 0.264 2509 214 KGRPEDLEFT 0.240 2510 131 RISANDFLGS 0.240 2511 1 MWIDIFPQDV 0.216 2512 296 VLLLLVLLTV 0.210 2513 268 RPKTSFNWFV 0.200 2514 265 KPSRPKTSFN 0.200 2515 65 HDKQETDVHF 0.200 2516 150 RGARGPELCS 0.200 2517 227 GNVYILTGKV 0.198 2518 154 GPELCSVQLA 0.180 2519 303 LTVFLLLVFY 0.180 2520 38 VVLDDENPLT 0.180 2521 312 YTIPGQISQV 0.180 2522 158 CSVQLARNGA 0.180 2523 143 LQLPDMVRGA 0.180 2524 295 LVLLLLVLLT 0.180 2525 244 TVEEAEKRPV 0.180 2526 75 NSLTGEGNFN 0.180 2527 158P3D2 v.2a A24- 10mers SEQ. ID Pos 1234567890 Score NO. 157 RYIPKQLNPI 216.000 2528 42 IYNRSLEEEF 198.000 2529 92 IYPESEAVLF 180.000 2530 45 RSLEEEFNHF 10.368 2531 122 VYVVKATNLA 9.000 2532 121 RVYVVKATNL 8.000 2533 166 IFGEILELSI 7.200 2534 73 GGEEEGSGHL 7.200 2535 162 QLNPIFGEIL 7.200 2536 164 NPIFGEILEL 6.600 2537 109 GIPQNRPIKL 6.600 2538 31 VSPKKAVATL 6.000 2539 38 ATLKIYNRSL 6.000 2540 110 IPQNRPIKLL 6.000 2541 183 LTVAVFEHDL 6.000 2542 197 DLIGETHIDL 6.000 2543 161 KQLNPIFGEI 5.544 2544 3 DPGDSDGVNL 4.800 2545 91 LIYPESEAVL 4.800 2546 174 SISLPAETEL 4.400 2547 211 YSHHRANCGL 4.000 2548 82 LVGKFKGSFL 4.000 2549 158 YIPKQLNPIF 3.600 2550 227 WVQQGPQEPF 3.000 2551 81 HLVGKFKGSF 3.000 2552 201 ETHIDLENRF 2.880 2553 77 EGSGHLVGKF 2.640 2554 101 FSEPQISRGI 2.520 2555 10 VNLISMVGEI 1.650 2556 97 EAVLFSEPQI 1.500 2557 223 QYEVWVQQGP 1.260 2558 209 RFYSHHRANC 1.000 2559 83 VGKFKGSFLI 1.000 2560 154 TKERYIPKQL 0.840 2561 89 SFLIYPESEA 0.825 2562 50 EFNHFEDWLN 0.750 2563 168 GEILELSISL 0.720 2564 63 LYRGQGGQDG 0.600 2565 210 FYSHHRANCG 0.600 2566 6 DSDGVNLISM 0.500 2567 189 EHDLVGSDDL 0.400 2568 49 EEFNHFEDWL 0.400 2569 54 FEDWLNVFPL 0.400 2570 114 RPIKLLVRVY 0.360 2571 215 RANCGLASQY 0.360 2572 35 KAVATLKIYN 0.360 2573 138 KADPYVVVSA 0.336 2574 87 KGSFLIYPES 0.308 2575 52 NHFEDWLNVF 0.288 2576 179 AETELTVAVF 0.240 2577 199 IGETHIDLEN 0.231 2578 22 QGEAEVKGTV 0.210 2579 170 ILELSISLPA 0.210 2580 28 KGTVSPKKAV 0.200 2581 18 EIQDQGEAEV 0.198 2582 150 ERQDTKERYI 0.180 2583 175 ISLPAETELT 0.180 2584 98 AVLFSEPQIS 0.180 2585 37 VATLKIYNRS 0.168 2586 130 LAPADPNGKA 0.165 2587 16 VGEIQDQGEA 0.165 2588 118 LLVRVYVVKA 0.165 2589 193 VGSDDLIGET 0.158 2590 167 FGEILELSIS 0.150 2591 90 FLIYPESEAV 0.150 2592 29 GTVSPKKAVA 0.150 2593 93 YPESEAVLFS 0.150 2594 190 HDLVGSDDLI 0.150 2595 218 CGLASQYEVW 0.150 2596 127 ATNLAPADPN 0.150 2597 176 SLPAETELTV 0.150 2598 134 DPNGKADPYV 0.150 2599 119 LVRVYVVKAT 0.140 2600 172 ELSISLPAET 0.132 2601 30 TVSPKKAVAT 0.120 2602 145 VSAGRERQDT 0.120 2603 4 PGDSDGVNLI 0.120 2604 21 DQGEAEVKGT 0.120 2605 186 AVFEHDLVGS 0.120 2606 177 LPAETELTVA 0.120 2607 217 NCGLASQYEV 0.110 2608 53 HFEDWLNVFP 0.108 2609 107 SRGIPQNRPI 0.100 2610 184 TVAVFEHDLV 0.100 2611 124 VVKATNLAPA 0.100 2612 207 ENRFYSHHRA 0.100 2613 203 HIDLENRFYS 0.100 2614 136 NGKADPYVVV 0.100 2615 51 FNHFEDWLNV 0.100 2616 85 KFKGSFLIYP 0.100 2617 195 SDDLIGETHI 0.100 2618 219 GLASQYEVWV 0.100 2619 43 YNRSLEEEFN 0.100 2620 60 VFPLYRGQGG 0.090 2621 187 VFEHDLVGSD 0.090 2622 141 PYVVVSAGRE 0.075 2623 100 LFSEPQISRG 0.060 2624 117 KLLVRVYVVK 0.042 2625 108 RGIPQNRPIK 0.036 2626 65 RGQGGQDGGG 0.030 2627 158P3D2 v.3 A24- 10mers SEQ. ID Pos 1234567890 Score NO. 7 VSVRRRSGPF 3.000 2628 9 VRRRSGPFAL 0.400 2629 8 SVRRRSGPFA 0.100 2630 4 EREVSVRRRS 0.021 2631 1 LPTEREVSVR 0.012 2632 6 EVSVRRRSGP 0.010 2633 5 REVSVRRRSG 0.003 2634 10 RRRSGPFALE 0.002 2635 2 PTEREVSVRR 0.002 2636 3 TEREVSVRRR 0.001 2637 158P3D2 v.4 A24- 10mers SEQ. ID Pos 1234567890 Score NO. 10 IWRRSGPFAL 4.000 2638 8 VSIWRRSGPF 3.000 2639 1 YLPTEREVSI 1.500 2640 2 LPTEREVSIW 0.120 2641 9 SIWRRSGPFA 0.100 2642 5 EREVSIWRRS 0.021 2643 7 EVSIWRRSGP 0.010 2644 6 REVSIWRRSG 0.003 2645 3 PTEREVSIWR 0.002 2646 4 TEREVSIWRR 0.001 2647 158P3D2 v.5a A24- 10mers SEQ. ID Pos 1234567890 Score NO. 8 DYTASLPMTS 6.000 2648 4 LQVWDYTASL 6.000 2649 30 VGPGAPSSAL 6.000 2650 9 YTASLPMTSL 4.000 2651 45 MGPGRGAICF 3.000 2652 24 SYQTWCVGPG 0.750 2653 36 SSALCSWPAM 0.500 2654 6 VWDYTASLPM 0.500 2655 1 VLVLQVWDYT 0.210 2656 49 RGAICFAAAA 0.200 2657 16 TSLDPWSCSY 0.180 2658 13 LPMTSLDPWS 0.180 2659 43 PAMGPGRGAI 0.150 2660 3 VLQVWDYTAS 0.150 2661 12 SLPMTSLDPW 0.150 2662 28 WCVGPGAPSS 0.150 2663 33 GAPSSALCSW 0.150 2664 2 LVLQVWDYTA 0.150 2665 25 YQTWCVGPGA 0.140 2666 19 DPWSCSYQTW 0.120 2667 29 CVGPGAPSSA 0.120 2668 44 AMGPGRGAIC 0.120 2669 27 TWCVGPGAPS 0.100 2670 42 WPAMGPGRGA 0.100 2671 15 MTSLDPWSCS 0.100 2672 46 GPGRGAICFA 0.100 2673 21 WSCSYQTWCV 0.100 2674 31 GPGAPSSALC 0.100 2675 11 ASLPMTSLDP 0.018 2676 18 LDPWSCSYQT 0.015 2677 37 SALCSWPAMG 0.015 2678 41 SWPAMGPGRG 0.015 2679 47 PGRGAICFAA 0.014 2680 34 APSSALCSWP 0.012 2681 40 CSWPAMGPGR 0.012 2682 48 GRGAICFAAA 0.012 2683 32 PGAPSSALCS 0.012 2684 17 SLDPWSCSYQ 0.012 2685 5 QVWDYTASLP 0.012 2686 7 WDYTASLPMT 0.010 2687 39 LCSWPAMGPG 0.010 2688 23 CSYQTWCVGP 0.010 2689 20 PWSCSYQTWC 0.010 2690 14 PMTSLDPWSC 0.010 2691 26 QTWCVGPGAP 0.010 2692 10 TASLPMTSLD 0.010 2693 35 PSSALCSWPA 0.010 2694 38 ALCSWPAMGP 0.010 2695 22 SCSYQTWCVG 0.010 2696

TABLE XV 158P3D2 B7, 9mers (variants 1, 2a, 3, 4 and 5a) 158P3D2 v.1 B7-9mers SEQ. ID Pos 123456789 Score NO. 255 KGRKQPEPL 40.000 2697 154 GPELCSVQL 24.000 2698 300 LVLLTVFLL 20.000 2699 55 YVKSWVKGL 20.000 2700 168 GPRCNLFRC 20.000 2701 295 LVLLLLVLL 20.000 2702 38 VVLDDENPL 20.000 2703 133 SANDFLGSL 12.000 2704 10 VPAPPPVDI 12.000 2705 165 NGAGPRCNL 9.000 2706 314 IPGQISQVI 8.000 2707 180 RGWWPVVKL 6.000 2708 235 KVEAEFELL 6.000 2709 294 TLVLLLLVL 4.000 2710 94 LPTEREVSV 4.000 2711 22 QPISYELRV 4.000 2712 301 VLLTVFLLL 4.000 2713 291 YWRTLVLLL 4.000 2714 318 ISQVIFRPL 4.000 2715 103 WRRSGPFAL 4.000 2716 299 LLVLLTVFL 4.000 2717 118 QPAVLVLQV 4.000 2718 137 FLGSLELQL 4.000 2719 287 IWRRYWRTL 4.000 2720 171 CNLFRCRRL 4.000 2721 224 DMGGNVYIL 4.000 2722 19 KPRQPISYE 3.000 2723 178 RLRGWWPVV 2.000 2724 183 WPVVKLKEA 2.000 2725 114 AEFRQPAVL 1.200 2726 69 ETDVHFNSL 1.200 2727 276 FVNPLKTFV 1.000 2728 27 ELRVVIWNT 1.000 2729 30 VVIWNTEDV 1.000 2730 228 NVYILTGKV 1.000 2731 151 GARGPELCS 0.900 2732 159 SVQLARNGA 0.750 2733 148 MVRGARGPE 0.750 2734 24 ISYELRVVI 0.600 2735 265 KPSRPKTSF 0.600 2736 12 APPPVDIKP 0.600 2737 292 WRTLVLLLL 0.400 2738 32 IWNTEDVVL 0.400 2739 289 RRYWRTLVL 0.400 2740 149 VRGARGPEL 0.400 2741 46 LTGEMSSDI 0.400 2742 306 FLLLVFYTI 0.400 2743 272 SFNWFVNPL 0.400 2744 234 GKVEAEFEL 0.400 2745 278 NPLKTFVFF 0.400 2746 130 DRISANDFL 0.400 2747 86 RFVFRFDYL 0.400 2748 135 NDFLGSLEL 0.400 2749 44 NPLTGEMSS 0.400 2750 212 RRKGRPEDL 0.400 2751 268 RPKTSFNWF 0.400 2752 290 RYWRTLVLL 0.400 2753 116 FRQPAVLVL 0.400 2754 124 LQVWDYDRI 0.400 2755 140 SLELQLPDM 0.300 2756 288 WRRYWRTLV 0.300 2757 162 LARNGAGPR 0.300 2758 115 EFRQPAVLV 0.300 2759 175 RCRRLRGWW 0.300 2760 214 KGRPEDLEF 0.200 2761 80 EGNFNWRFV 0.200 2762 302 LLTVFLLLV 0.200 2763 297 LLLLVLLTV 0.200 2764 261 EPLEKPSRP 0.200 2765 107 GPFALEEAE 0.200 2766 31 VIWNTEDVV 0.200 2767 313 TIPGQISQV 0.200 2768 251 RPVGKGRKQ 0.200 2769 293 RTLVLLLLV 0.200 2770 6 FPQDVPAPP 0.200 2771 237 EAEFELLTV 0.180 2772 113 EAEFRQPAV 0.180 2773 193 DVEREAQEA 0.150 2774 120 AVLVLQVWD 0.150 2775 259 QPEPLEKPS 0.120 2776 223 TDMGGNVYI 0.120 2777 283 FVFFIWRRY 0.100 2778 106 SGPFALEEA 0.100 2779 101 SVWRRSGPF 0.100 2780 150 RGARGPELC 0.100 2781 304 TVFLLLVFY 0.100 2782 42 DENPLTGEM 0.100 2783 296 VLLLLVLLT 0.100 2784 125 QVWDYDRIS 0.100 2785 225 MGGNVYILT 0.100 2786 144 QLPDMVRGA 0.100 2787 102 VWRRSGPFA 0.100 2788 4 DIFPQDVPA 0.100 2789 88 VFRFDYLPT 0.100 2790 209 KQRRRKGRP 0.100 2791 286 FIWRRYWRT 0.100 2792 230 YILTGKVEA 0.100 2793 16 VDIKPRQPI 0.090 2794 145 LPDMVRGAR 0.090 2795 190 EAEDVEREA 0.090 2796 158P3D2 v.2a B7-9-mers SEQ. ID Pos 123456789 Score NO. 32 SPKKAVATL 80.000 2797 110 IPQNRPIKL 80.000 2798 184 TVAVFEHDL 20.000 2799 131 APADPNGKA 9.000 2800 98 AVLFSEPQI 6.000 2801 119 LVRVYVVKA 5.000 2802 198 LIGETHIDL 4.000 2803 175 ISLPAETEL 4.000 2804 169 EILELSISL 4.000 2805 114 RPIKLLVRV 4.000 2806 83 VGKFKGSFL 4.000 2807 163 LNPIFGEIL 4.000 2808 39 TLKIYNRSL 4.000 2809 177 LPAETELTV 4.000 2810 155 KERYIPKQL 4.000 2811 112 QNRPIKLLV 2.000 2812 220 LASQYEVWV 0.600 2813 111 PQNRPIKLL 0.600 2814 185 VAVFEHDLV 0.600 2815 123 YVVKATNLA 0.500 2816 30 TVSPKKAVA 0.500 2817 146 SAGRERQDT 0.450 2818 190 HDLVGSDDL 0.400 2819 92 IYPESEAVL 0.400 2820 212 SHHRANCGL 0.400 2821 122 VYVVKATNL 0.400 2822 55 EDWLNVFPL 0.400 2823 191 DLVGSDDLI 0.400 2824 165 PIFGEILEL 0.400 2825 50 EFNHFEDWL 0.400 2826 159 IPKQLNPIF 0.400 2827 108 RGIPQNRPI 0.400 2828 162 QLNPIFGEI 0.400 2829 134 DPNGKADPY 0.400 2830 3 DPGDSDGVN 0.400 2831 11 NLISMVGEI 0.400 2832 158 YIPKQLNPI 0.400 2833 29 GTVSPKKAV 0.300 2834 103 EPQISRGIP 0.300 2835 147 AGRERQDTK 0.300 2836 36 AVATLKIYN 0.300 2837 61 FPLYRGQGG 0.200 2838 164 NPIFGEILE 0.200 2839 136 NGKADPYVV 0.200 2840 8 DGVNLISMV 0.200 2841 218 CGLASQYEV 0.200 2842 43 YNRSLEEEF 0.200 2843 117 KLLVRVYVV 0.200 2844 91 LIYPESEAV 0.200 2845 140 DPYVVVSAG 0.200 2846 186 AVFEHDLVG 0.150 2847 90 FLIYPESEA 0.150 2848 93 YPESEAVLF 0.120 2849 74 GEEEGSGHL 0.120 2850 167 FGEILELSI 0.120 2851 151 RQDTKERYI 0.120 2852 4 PGDSDGVNL 0.120 2853 207 ENRFYSHHR 0.100 2854 28 KGTVSPKKA 0.100 2855 213 HHRANCGLA 0.100 2856 173 LSISLPAET 0.100 2857 176 SLPAETELT 0.100 2858 7 SDGVNLISM 0.100 2859 31 VSPKKAVAT 0.100 2860 121 RVYVVKATN 0.100 2861 106 ISRGIPQNR 0.100 2862 82 LVGKFKGSF 0.100 2863 144 VVSAGRERQ 0.075 2864 38 ATLKIYNRS 0.060 2865 179 AETELTVAV 0.060 2866 216 ANCGLASQY 0.060 2867 35 KAVATLKIY 0.060 2868 19 IQDQGEAEV 0.060 2869 143 VVVSAGRER 0.050 2870 192 LVGSDDLIG 0.050 2871 142 YVVVSAGRE 0.050 2872 9 GVNLISMVG 0.050 2873 124 VVKATNLAP 0.050 2874 59 NVFPLYRGQ 0.050 2875 26 EVKGTVSPK 0.050 2876 225 EVWVQQGPQ 0.050 2877 227 WVQQGPQEP 0.050 2878 15 MVGEIQDQG 0.050 2879 34 KKAVATLKI 0.040 2880 5 GDSDGVNLI 0.040 2881 196 DDLIGETHI 0.040 2882 84 GKFKGSFLI 0.040 2883 102 SEPQISRGI 0.040 2884 116 IKLLVRVYV 0.030 2885 128 TNLAPADPN 0.030 2886 126 KATNLAPAD 0.030 2887 221 ASQYEVWVQ 0.030 2888 130 LAPADPNGK 0.030 2889 228 VQQGPQEPF 0.030 2890 37 VATLKIYNR 0.030 2891 137 GKADPYVVV 0.030 2892 139 ADPYVVVSA 0.030 2893 13 ISMVGEIQD 0.030 2894 215 RANCGLASQ 0.030 2895 127 ATNLAPADP 0.030 2896 158P3D2 v.3 B7-9mers SEQ. ID Pos 123456789 Score NO. 9 RRRSGPFAL 4.000 2897 7 SVRRRSGPF 1.000 2898 8 VRRRSGPFA 0.100 2899 5 EVSVRRRSG 0.075 2900 6 VSVRRRSGP 0.015 2901 2 TEREVSVRR 0.010 2902 4 REVSVRRRS 0.003 2903 3 EREVSVRRR 0.000 2904 1 PTEREVSVR 0.000 2905 158P3D2 v.4 B7-9mers SEQ. ID Pos 123456789 Score NO. 1 LPTEREVSI 8.000 2906 9 IWRRSGPFA 0.100 2907 6 EVSIWRRSG 0.075 2908 8 SIWRRSGPF 0.020 2909 7 VSIWRRSGP 0.015 2910 3 TEREVSIWR 0.010 2911 5 REVSIWRRS 0.002 2912 2 PTEREVSIW 0.001 2913 4 EREVSIWRR 0.000 2914 158P3D2 v.5a-B7-9-mers SEQ. ID Pos 123456789 Score NO. 30 GPGAPSSAL 120.000 2915 4 QVWDYTASL 20.000 2916 9 TASLPMTSL 18.000 2917 36 SALCSWPAM 3.000 2918 18 DPWSCSYQT 2.000 2919 43 AMGPGRGAI 1.800 2920 12 LPMTSLDPW 1.200 2921 33 APSSALCSW 1.200 2922 1 LVLQVWDYT 0.500 2923 45 GPGRGAICF 0.400 2924 49 GAICFAAAA 0.300 2925 41 WPAMGPGRG 0.200 2926 21 SCSYQTWCV 0.200 2927 42 PAMGPGRGA 0.135 2928 48 RGAICFAAA 0.100 2929 6 WDYTASLPM 0.100 2930 2 VLQVWDYTA 0.100 2931 35 SSALCSWPA 0.100 2932 28 CVGPGAPSS 0.100 2933 46 PGRGAICFA 0.100 2934 20 WSCSYQTWC 0.100 2935 29 VGPGAPSSA 0.100 2936 25 QTWCVGPGA 0.100 2937 14 MTSLDPWSC 0.100 2938 44 MGPGRGAIC 0.100 2939 32 GAPSSALCS 0.060 2940 15 TSLDPWSCS 0.030 2941 27 WCVGPGAPS 0.030 2942 10 ASLPMTSLD 0.030 2943 37 ALCSWPAMG 0.030 2944 8 YTASLPMTS 0.020 2945 3 LQVWDYTAS 0.020 2946 38 LCSWPAMGP 0.015 2947 11 SLPMTSLDP 0.010 2948 31 PGAPSSALC 0.010 2949 22 CSYQTWCVG 0.010 2950 39 CSWPAMGPG 0.010 2951 47 GRGAICFAA 0.010 2952 24 YQTWCVGPG 0.010 2953 7 DYTASLPMT 0.010 2954 16 SLDPWSCSY 0.006 2955 13 PMTSLDPWS 0.002 2956 17 LDPWSCSYQ 0.001 2957 40 SWPAMGPGR 0.001 2958 23 SYQTWCVGP 0.001 2959 26 TWCVGPGAP 0.001 2960 34 PSSALCSWP 0.001 2961 5 VWDYTASLP 0.000 2962 19 PWSCSYQTW 0.000 2963

TABLE XVI 158P3D2 B7, 10mers (variants 1, 2a, 3, 4 and 5a) 158P3D2 v.1 B7-10mers Pos 1234567890 Score SEQ. ID NO. 19 KPRQPISYEL 800.000 2964 148 MVRGARGPEL 200.000 2965 37 DVVLDDENPL 20.000 2966 300 LVLLTVFLLL 20.000 2967 164 RNGAGPRCNL 9.000 2968 278 NPLKTFVFFI 8.000 2969 151 GARGPELCSV 6.000 2970 216 RPEDLEFTDM 6.000 2971 31 VIWNTEDVVL 4.000 2972 298 LLLVLLTVFL 4.000 2973 294 TLVLLLLVLL 4.000 2974 129 YDRISANDFL 4.000 2975 132 ISANDFLGSL 4.000 2976 288 WRRYWRTLVL 4.000 2977 170 RCNLFRCRRL 4.000 2978 22 QPISYELRVV 4.000 2979 115 EFRQPAVLVL 4.000 2980 153 RGPELCSVQL 4.000 2981 293 RTLVLLLLVL 4.000 2982 291 YWRTLVLLLL 4.000 2983 286 FIWRRYWRTL 4.000 2984 271 TSFNWFVNPL 4.000 2985 233 TGKVEAEFEL 4.000 2986 268 RPKTSFNWFV 4.000 2987 299 LLVLLTVFLL 4.000 2988 211 RRRKGRPEDL 4.000 2989 102 VWRRSGPFAL 4.000 2990 317 QISQVIFRPL 4.000 2991 134 ANDFLGSLEL 3.600 2992 113 EAEFRQPAVL 3.600 2993 9 DVPAPPPVDI 3.000 2994 162 LARNGAGPRC 3.000 2995 309 LVFYTIPGQI 2.000 2996 168 GPRCNLFRCR 2.000 2997 223 TDMGGNVYIL 1.200 2998 30 VVIWNTEDVV 1.000 2999 29 RVVIWNTEDV 1.000 3000 214 KGRPEDLEFT 1.000 3001 185 VVKLKEAEDV 1.000 3002 139 GSLELQLPDM 1.000 3003 125 QVWDYDRISA 0.750 3004 12 APPPVDIKPR 0.600 3005 154 GPELCSVQLA 0.600 3006 179 LRGWWPVVKL 0.600 3007 295 LVLLLLVLLT 0.500 3008 38 VVLDDENPLT 0.500 3009 87 FVFRFDYLPT 0.500 3010 101 SVWRRSGPFA 0.500 3011 304 TVFLLLVFYT 0.500 3012 54 IYVKSWVKGL 0.400 3013 313 TIPGQISQVI 0.400 3014 289 RRYWRTLVLL 0.400 3015 136 DFLGSLELQL 0.400 3016 234 GKVEAEFELL 0.400 3017 254 GKGRKQPEPL 0.400 3018 118 QPAVLVLQVW 0.400 3019 314 IPGQISQVIF 0.400 3020 68 QETDVHFNSL 0.400 3021 107 GPFALEEAEF 0.400 3022 123 VLQVWDYDRI 0.400 3023 290 RYWRTLVLLL 0.400 3024 94 LPTEREVSVW 0.400 3025 265 KPSRPKTSFN 0.400 3026 85 WRFVFRFDYL 0.400 3027 261 EPLEKPSRPK 0.300 3028 10 VPAPPPVDIK 0.300 3029 120 AVLVLQVWDY 0.300 3030 167 AGPRCNLFRC 0.300 3031 287 IWRRYWRTLV 0.300 3032 244 TVEEAEKRPV 0.300 3033 251 RPVGKGRKQP 0.300 3034 6 FPQDVPAPPP 0.300 3035 296 VLLLLVLLTV 0.200 3036 117 RQPAVLVLQV 0.200 3037 44 NPLTGEMSSD 0.200 3038 176 CRRLRGWWPV 0.200 3039 183 WPVVKLKEAE 0.200 3040 301 VLLTVFLLLV 0.200 3041 227 GNVYILTGKV 0.200 3042 21 RQPISYELRV 0.200 3043 312 YTIPGQISQV 0.200 3044 93 YLPTEREVSV 0.200 3045 235 KVEAEFELLT 0.150 3046 158 CSVQLARNGA 0.150 3047 283 FVFFIWRRYW 0.150 3048 255 KGRKQPEPLE 0.150 3049 15 PVDIKPRQPI 0.135 3050 222 FTDMGGNVYI 0.120 3051 209 KQRRRKGRPE 0.100 3052 105 RSGPFALEEA 0.100 3053 27 ELRVVIWNTE 0.100 3054 273 FNWFVNPLKT 0.100 3055 143 LQLPDMVRGA 0.100 3056 175 RCRRLRGWWP 0.100 3057 276 FVNPLKTFVF 0.100 3058 61 KGLEHDKQET 0.100 3059 224 DMGGNVYILT 0.100 3060 178 RLRGWWPVVK 0.100 3061 194 VEREAQEAQA 0.100 3062 114 AEFRQPAVLV 0.090 3063 158P392 v.2a B7-10mers Pos 1234567890 Score SEQ. ID NO. 110 IPQNRPIKLL 120.000 3064 164 NPIFGEILEL 80.000 3065 3 DPGDSDGVNL 80.000 3066 121 RVYVVKATNL 20.000 3067 82 LVGKFKGSFL 20.000 3068 38 ATLKIYNRSL 12.000 3069 119 LVRVYVVKAT 5.000 3070 91 LIYPESEAVL 4.000 3071 197 DLIGETHIDL 4.000 3072 211 YSHHRANCGL 4.000 3073 31 VSPKKAVATL 4.000 3074 162 QLNPIFGEIL 4.000 3075 174 SISLPAETEL 4.000 3076 134 DPNGKADPYV 4.000 3077 109 GIPQNRPIKL 4.000 3078 183 LTVAVFEHDL 4.000 3079 177 LPAETELTVA 2.000 3080 73 GGEEEGSGHL 1.200 3081 97 EAVLFSEPQI 1.200 3082 184 TVAVFEHDLV 1.000 3083 207 ENRFYSHHRA 1.000 3084 131 APADPNGKAD 0.600 3085 124 VVKATNLAPA 0.500 3086 30 TVSPKKAVAT 0.500 3087 130 LAPADPNGKA 0.450 3088 83 VGKFKGSFLI 0.400 3089 161 KQLNPIFGEI 0.400 3090 10 VNLISMVGEI 0.400 3091 114 RPIKLLVRVY 0.400 3092 168 GEILELSISL 0.400 3093 49 EEFNHFEDWL 0.400 3094 98 AVLFSEPQIS 0.300 3095 147 AGRERQDTKE 0.300 3096 136 NGKADPYVVV 0.300 3097 6 DSDGVNLISM 0.300 3098 186 AVFEHDLVGS 0.300 3099 28 KGTVSPKKAV 0.300 3100 32 SPKKAVATLK 0.200 3101 219 GLASQYEVWV 0.200 3102 61 FPLYRGQGGQ 0.200 3103 18 EIQDQGEAEV 0.200 3104 217 NCGLASQYEV 0.200 3105 103 EPQISRGIPQ 0.200 3106 51 FNHFEDWLNV 0.200 3107 140 DPYVVVSAGR 0.200 3108 90 FLIYPESEAV 0.200 3109 159 IPKQLNPIFG 0.200 3110 176 SLPAETELTV 0.200 3111 43 YNRSLEEEFN 0.200 3112 106 ISRGIPQNRP 0.150 3113 36 AVATLKIYNR 0.150 3114 145 VSAGRERQDT 0.150 3115 227 WVQQGPQEPF 0.150 3116 93 YPESEAVLFS 0.120 3117 154 TKERYIPKQL 0.120 3118 189 EHDLVGSDDL 0.120 3119 54 FEDWLNVFPL 0.120 3120 101 FSEPQISRGI 0.120 3121 29 GTVSPKKAVA 0.100 3122 112 QNRPIKLLVR 0.100 3123 175 ISLPAETELT 0.100 3124 21 DQGEAEVKGT 0.100 3125 193 VGSDDLIGET 0.100 3126 118 LLVRVYVVKA 0.100 3127 172 ELSISLPAET 0.100 3128 127 ATNLAPADPN 0.090 3129 138 KADPYVVVSA 0.090 3130 143 VVVSAGRERQ 0.075 3131 59 NVFPLYRGQG 0.075 3132 35 KAVATLKIYN 0.060 3133 215 RANCGLASQY 0.060 3134 37 VATLKIYNRS 0.060 3135 22 QGEAEVKGTV 0.060 3136 192 LVGSDDLIGE 0.050 3137 142 YVVVSAGRER 0.050 3138 144 VVSAGRERQD 0.050 3139 9 GVNLISMVGE 0.050 3140 225 EVWVQQGPQE 0.050 3141 123 YVVKATNLAP 0.050 3142 26 EVKGTVSPKK 0.050 3143 15 MVGEIQDQGE 0.050 3144 107 SRGIPQNRPI 0.040 3145 166 IFGEILELSI 0.040 3146 157 RYIPKQLNPI 0.040 3147 190 HDLVGSDDLI 0.040 3148 150 ERQDTKERYI 0.040 3149 220 LASQYEVWVQ 0.030 3150 216 ANCGLASQYE 0.030 3151 126 KATNLAPADP 0.030 3152 146 SAGRERQDTK 0.030 3153 13 ISMVGEIQDQ 0.030 3154 221 ASQYEVWVQQ 0.030 3155 185 VAVFEHDLVG 0.030 3156 115 PIKLLVRVYV 0.030 3157 155 KERYIPKQLN 0.030 3158 16 VGEIQDQGEA 0.030 3159 170 ILELSISLPA 0.030 3160 158 YIPKQLNPIF 0.020 3161 7 SDGVNLISMV 0.020 3162 149 RERQDTKERY 0.020 3163 158P3D2 v.3 B7-10mers Pos 1234567890 Score SEQ. ID NO. 8 SVRRRSGPFA 5.000 3164 9 VRRRSGPFAL 4.000 3165 1 LPTEREVSVR 0.200 3166 6 EVSVRRRSGP 0.075 3167 7 VSVRRRSGPF 0.020 3168 10 RRRSGPFALE 0.015 3169 3 TEREVSVRRR 0.010 3170 5 REVSVRRRSG 0.002 3171 4 EREVSVRRRS 0.001 3172 2 PTEREVSVRR 0.000 3173 158P3D2 v.4 B7- 10mers Pos 1234567890 Score SEQ. ID NO. 10 IWRRSGPFAL 4.000 3174 1 YLPTEREVSI 0.400 3175 2 LPTEREVSIW 0.400 3176 9 SIWRRSGPFA 0.100 3177 7 EVSIWRRSGP 0.075 3178 8 VSIWRRSGPF 0.020 3179 4 TEREVSIWRR 0.010 3180 6 REVSIWRRSG 0.002 3181 5 EREVSIWRRS 0.001 3182 3 PTEREVSIWR 0.000 3183 158P3D2 v.5a B7-10mers Pos 1234567890 Score SEQ. ID NO. 9 YTASLPMTSL 6.000 3184 30 VGPGAPSSAL 6.000 3185 4 LQVWDYTASL 4.000 3186 42 WPAMGPGRGA 3.000 3187 46 GPGRGAICFA 2.000 3188 31 GPGAPSSALC 2.000 3189 13 LPMTSLDPWS 1.200 3190 36 SSALCSWPAM 1.000 3191 34 APSSALCSWP 0.600 3192 43 PAMGPGRGAI 0.540 3193 29 CVGPGAPSSA 0.500 3194 2 LVLQVWDYTA 0.500 3195 19 DPWSCSYQTW 0.400 3196 44 AMGPGRGAIC 0.300 3197 21 WSCSYQTWCV 0.200 3198 1 VLVLQVWDYT 0.100 3199 25 YQTWCVGPGA 0.100 3200 49 RGAICFAAAA 0.100 3201 47 PGRGAICFAA 0.100 3202 33 GAPSSALCSW 0.060 3203 5 QVWDYTASLP 0.050 3204 38 ALCSWPAMGP 0.045 3205 15 MTSLDPWSCS 0.030 3206 11 ASLPMTSLDP 0.030 3207 10 TASLPMTSLD 0.030 3208 37 SALCSWPAMG 0.030 3209 6 VWDYTASLPM 0.030 3210 45 MGPGRGAICF 0.020 3211 16 TSLDPWSCSY 0.020 3212 12 SLPMTSLDPW 0.020 3213 28 WCVGPGAPSS 0.020 3214 3 VLQVWDYTAS 0.020 3215 48 GRGAICFAAA 0.010 3216 22 SCSYQTWCVG 0.010 3217 23 CSYQTWCVGP 0.010 3218 18 LDPWSCSYQT 0.010 3219 14 PMTSLDPWSC 0.010 3220 7 WDYTASLPMT 0.010 3221 35 PSSALCSWPA 0.010 3222 39 LCSWPAMGPG 0.010 3223 26 QTWCVGPGAP 0.010 3224 40 CSWPAMGPGR 0.010 3225 27 TWCVGPGAPS 0.003 3226 17 SLDPWSCSYQ 0.003 3227 8 DYTASLPMTS 0.002 3228 32 PGAPSSALCS 0.002 3229 24 SYQTWCVGPG 0.001 3230 41 SWPAMGPGRG 0.001 3231 20 PWSCSYQTWC 0.001 3232

TABLE XVII 158P3D2 B35, 9mers (variants 1, 2a, 3, 4 and 5a) 158P3D2 v.1 B35-9mers Pos 123456789 Score SEQ. ID NO. 268 RPKTSFNWF 120.000 3233 265 KPSRPKTSF 40.000 3234 278 NPLKTFVFF 20.000 3235 214 KGRPEDLEF 9.000 3236 314 IPGQISQVI 8.000 3237 94 LPTEREVSV 8.000 3238 10 VPAPPPVDI 8.000 3239 154 GPELCSVQL 6.000 3240 168 GPRCNLFRC 6.000 3241 255 KGRKQPEPL 6.000 3242 133 SANDFLGSL 6.000 3243 318 ISQVIFRPL 5.000 3244 75 NSLTGEGNF 5.000 3245 118 QPAVLVLQV 4.000 3246 24 ISYELRVVI 4.000 3247 22 QPISYELRV 4.000 3248 38 VVLDDENPL 3.000 3249 55 YVKSWVKGL 3.000 3250 175 RCRRLRGWW 3.000 3251 166 GAGPRCNLF 3.000 3252 180 RGWWPVVKL 2.000 3253 183 WPVVKLKEA 2.000 3254 283 FVFFIWRRY 2.000 3255 304 TVFLLLVFY 2.000 3256 121 VLVLQVWDY 2.000 3257 44 NPLTGEMSS 2.000 3258 19 KPRQPISYE 1.200 3259 178 RLRGWWPVV 1.200 3260 299 LLVLLTVFL 1.000 3261 165 NGAGPRCNL 1.000 3262 224 DMGGNVYIL 1.000 3263 277 VNPLKTFVF 1.000 3264 298 LLLVLLTVF 1.000 3265 294 TLVLLLLVL 1.000 3266 137 FLGSLELQL 1.000 3267 171 CNLFRCRRL 1.000 3268 101 SVWRRSGPF 1.000 3269 81 GNFNWRFVF 1.000 3270 300 LVLLTVFLL 1.000 3271 50 MSSDIYVKS 1.000 3272 83 FNWRFVFRF 1.000 3273 232 LTGKVEAEF 1.000 3274 303 LTVFLLLVF 1.000 3275 301 VLLTVFLLL 1.000 3276 295 LVLLLLVLL 1.000 3277 77 LTGEGNFNW 1.000 3278 235 KVEAEFELL 0.900 3279 151 GARGPELCS 0.900 3280 46 LTGEMSSDI 0.800 3281 51 SSDIYVKSW 0.750 3282 132 ISANDFLGS 0.750 3283 222 FTDMGGNVY 0.600 3284 47 TGEMSSDIY 0.600 3285 259 QPEPLEKPS 0.600 3286 140 SLELQLPDM 0.600 3287 212 RRKGRPEDL 0.600 3288 124 LQVWDYDRI 0.600 3289 293 RTLVLLLLV 0.400 3290 306 FLLLVFYTI 0.400 3291 251 RPVGKGRKQ 0.400 3292 6 FPQDVPAPP 0.400 3293 261 EPLEKPSRP 0.400 3294 129 YDRISANDF 0.300 3295 291 YWRTLVLLL 0.300 3296 17 DIKPRQPIS 0.300 3297 27 ELRVVIWNT 0.300 3298 287 IWRRYWRTL 0.300 3299 69 ETDVHFNSL 0.300 3300 103 WRRSGPFAL 0.300 3301 237 EAEFELLTV 0.270 3302 216 RPEDLEFTD 0.240 3303 164 RNGAGPRCN 0.200 3304 234 GKVEAEFEL 0.200 3305 30 VVIWNTEDV 0.200 3306 313 TIPGQISQV 0.200 3307 18 IKPRQPISY 0.200 3308 150 RGARGPELC 0.200 3309 297 LLLLVLLTV 0.200 3310 42 DENPLTGEM 0.200 3311 107 GPFALEEAE 0.200 3312 290 RYWRTLVLL 0.200 3313 302 LLTVFLLLV 0.200 3314 12 APPPVDIKP 0.200 3315 31 VIWNTEDVV 0.200 3316 276 FVNPLKTFV 0.200 3317 228 NVYILTGKV 0.200 3318 125 QVWDYDRIS 0.200 3319 86 RFVFRFDYL 0.200 3320 144 QLPDMVRGA 0.200 3321 66 DKQETDVHF 0.200 3322 80 EGNFNWRFV 0.200 3323 85 WRFVFRFDY 0.200 3324 289 RRYWRTLVL 0.200 3325 270 KTSFNWFVN 0.200 3326 113 EAEFRQPAV 0.180 3327 190 EAEDVEREA 0.180 3328 76 SLTGEGNFN 0.150 3329 266 PSRPKTSFN 0.150 3330 32 IWNTEDVVL 0.150 3331 119 PAVLVLQVW 0.150 3332 158P3D2 v.2a B35-9-mers Pos 123456789 Score SEQ. ID NO. 32 SPKKAVATL 60.000 3333 159 IPKQLNPIF 60.000 3334 134 DPNGKADPY 40.000 3335 110 IPQNRPIKL 20.000 3336 35 KAVATLKIY 12.000 3337 93 YPESEAVLF 9.000 3338 177 LPAETELTV 8.000 3339 114 RPIKLLVRV 8.000 3340 78 GSGHLVGKF 5.000 3341 175 ISLPAETEL 5.000 3342 131 APADPNGKA 4.000 3343 3 DPGDSDGVN 4.000 3344 39 TLKIYNRSL 3.000 3345 83 VGKFKGSFL 3.000 3346 43 YNRSLEEEF 3.000 3347 198 LIGETHIDL 2.000 3348 216 ANCGLASQY 2.000 3349 169 EILELSISL 2.000 3350 85 KFKGSFLIY 1.200 3351 228 VQQGPQEPF 1.000 3352 184 TVAVFEHDL 1.000 3353 82 LVGKFKGSF 1.000 3354 163 LNPIFGEIL 1.000 3355 136 NGKADPYVV 0.900 3356 203 HIDLENRFY 0.900 3357 185 VAVFEHDLV 0.900 3358 46 SLEEEFNHF 0.900 3359 108 RGIPQNRPI 0.800 3360 155 KERYIPKQL 0.600 3361 112 QNRPIKLLV 0.600 3362 220 LASQYEVWV 0.600 3363 115 PIKLLVRVY 0.600 3364 88 GSFLIYPES 0.500 3365 219 GLASQYEVW 0.500 3366 173 LSISLPAET 0.500 3367 31 VSPKKAVAT 0.500 3368 146 SAGRERQDT 0.450 3369 158 YIPKQLNPI 0.400 3370 11 NLISMVGEI 0.400 3371 191 DLVGSDDLI 0.400 3372 98 AVLFSEPQI 0.400 3373 117 KLLVRVYVV 0.400 3374 162 QLNPIFGEI 0.400 3375 150 ERQDTKERY 0.400 3376 194 GSDDLIGET 0.300 3377 91 LIYPESEAV 0.300 3378 119 LVRVYVVKA 0.300 3379 45 RSLEEEFNH 0.300 3380 180 ETELTVAVF 0.300 3381 151 RQDTKERYI 0.240 3382 140 DPYVVVSAG 0.200 3383 121 RVYVVKATN 0.200 3384 202 THIDLENRF 0.200 3385 28 KGTVSPKKA 0.200 3386 7 SDGVNLISM 0.200 3387 56 DWLNVFPLY 0.200 3388 8 DGVNLISMV 0.200 3389 164 NPIFGEILE 0.200 3390 218 CGLASQYEV 0.200 3391 29 GTVSPKKAV 0.200 3392 61 FPLYRGQGG 0.200 3393 92 IYPESEAVL 0.200 3394 103 EPQISRGIP 0.200 3395 138 KADPYVVVS 0.180 3396 165 PIFGEILEL 0.150 3397 106 ISRGIPQNR 0.150 3398 176 SLPAETELT 0.150 3399 6 DSDGVNLIS 0.150 3400 51 FNHFEDWLN 0.150 3401 99 VLFSEPQIS 0.150 3402 71 DGGGEEEGS 0.150 3403 167 FGEILELSI 0.120 3404 212 SHHRANCGL 0.100 3405 50 EFNHFEDWL 0.100 3406 36 AVATLKIYN 0.100 3407 90 FLIYPESEA 0.100 3408 81 HLVGKFKGS 0.100 3409 122 VYVVKATNL 0.100 3410 105 QISRGIPQN 0.100 3411 190 HDLVGSDDL 0.100 3412 38 ATLKIYNRS 0.100 3413 123 YVVKATNLA 0.100 3414 111 PQNRPIKLL 0.100 3415 128 TNLAPADPN 0.100 3416 55 EDWLNVFPL 0.100 3417 30 TVSPKKAVA 0.100 3418 24 EAEVKGTVS 0.090 3419 34 KKAVATLKI 0.080 3420 5 GDSDGVNLI 0.080 3421 221 ASQYEVWVQ 0.075 3422 52 NHFEDWLNV 0.060 3423 126 KATNLAPAD 0.060 3424 215 RANCGLASQ 0.060 3425 153 DTKERYIPK 0.060 3426 147 AGRERQDTK 0.060 3427 53 HFEDWLNVF 0.060 3428 19 IQDQGEAEV 0.060 3429 74 GEEEGSGHL 0.060 3430 49 EEFNHFEDW 0.050 3431 145 VSAGRERQD 0.050 3432 158P3D2 v.3 B35-9-mers Pos 123456789 Score SEQ. ID NO. 7 SVRRRSGPF 3.000 3433 9 RRRSGPFAL 0.600 3434 6 VSVRRRSGP 0.050 3435 8 VRRRSGPFA 0.030 3436 4 REVSVRRRS 0.020 3437 5 EVSVRRRSG 0.010 3438 2 TEREVSVRR 0.006 3439 1 PTEREVSVR 0.000 3440 3 EREVSVRRR 0.000 3441 158P3D2 v.4 B35-9mers Pos 123456789 Score SEQ. ID NO. 1 LPTEREVSI 16.000 3442 8 SIWRRSGPF 1.000 3443 7 VSIWRRSGP 0.050 3444 9 IWRRSGPFA 0.030 3445 2 PTEREVSIW 0.022 3446 5 REVSIWRRS 0.020 3447 6 EVSIWRRSG 0.010 3448 3 TEREVSIWR 0.006 3449 4 EREVSIWRR 0.000 3450 158P3D2 v.5a B35-9mers Pos 123456789 Score SEQ. ID NO. 45 GPGRGAICF 20.000 3451 30 GPGAPSSAL 20.000 3452 33 APSSALCSW 10.000 3453 12 LPMTSLDPW 10.000 3454 36 SALCSWPAM 6.000 3455 9 TASLPMTSL 3.000 3456 4 QVWDYTASL 2.000 3457 18 DPWSCSYQT 2.000 3458 15 TSLDPWSCS 1.000 3459 16 SLDPWSCSY 0.600 3460 20 WSCSYQTWC 0.500 3461 35 SSALCSWPA 0.500 3462 43 AMGPGRGAI 0.400 3463 49 GAICFAAAA 0.300 3464 32 GAPSSALCS 0.300 3465 6 WDYTASLPM 0.200 3466 41 WPAMGPGRG 0.200 3467 21 SCSYQTWCV 0.200 3468 48 RGAICFAAA 0.200 3469 3 LQVWDYTAS 0.150 3470 14 MTSLDPWSC 0.150 3471 1 LVLQVWDYT 0.100 3472 2 VLQVWDYTA 0.100 3473 27 WCVGPGAPS 0.100 3474 25 QTWCVGPGA 0.100 3475 29 VGPGAPSSA 0.100 3476 44 MGPGRGAIC 0.100 3477 28 CVGPGAPSS 0.100 3478 8 YTASLPMTS 0.100 3479 10 ASLPMTSLD 0.050 3480 22 CSYQTWCVG 0.050 3481 39 CSWPAMGPG 0.050 3482 42 PAMGPGRGA 0.030 3483 46 PGRGAICFA 0.030 3484 13 PMTSLDPWS 0.010 3485 37 ALCSWPAMG 0.010 3486 38 LCSWPAMGP 0.010 3487 7 DYTASLPMT 0.010 3488 11 SLPMTSLDP 0.010 3489 24 YQTWCVGPG 0.010 3490 31 PGAPSSALC 0.010 3491 47 GRGAICFAA 0.010 3492 34 PSSALCSWP 0.005 3493 19 PWSCSYQTW 0.005 3494 17 LDPWSCSYQ 0.001 3495 26 TWCVGPGAP 0.001 3496 23 SYQTWCVGP 0.001 3497 40 SWPAMGPGR 0.001 3498 5 VWDYTASLP 0.000 3499

TABLE XVIII 158P3D2 B35, 10mers (variants 1, 2a, 3, 4 and 5a) 158P3D2 v.1 B35-10mers Pos 1234567890 Score SEQ. ID NO. 19 KPRQPISYEL 120.000 3500 216 RPEDLEFTDM 72.000 3501 94 LPTEREVSVW 30.000 3502 107 GPFALEEAEF 30.000 3503 268 RPKTSFNWFV 24.000 3504 139 GSLELQLPDM 20.000 3505 314 IPGQISQVIF 20.000 3506 118 QPAVLVLQVW 10.000 3507 278 NPLKTFVFFI 8.000 3508 17 DIKPRQPISY 6.000 3509 22 QPISYELRVV 6.000 3510 271 TSFNWFVNPL 5.000 3511 24 ISYELRVVIW 5.000 3512 50 MSSDIYVKSW 5.000 3513 132 ISANDFLGSL 5.000 3514 100 VSVWRRSGPF 5.000 3515 46 LTGEMSSDIY 4.000 3516 153 RGPELCSVQL 4.000 3517 265 KPSRPKTSFN 4.000 3518 148 MVRGARGPEL 3.000 3519 233 TGKVEAEFEL 3.000 3520 151 GARGPELCSV 2.700 3521 164 RNGAGPRCNL 2.000 3522 120 AVLVLQVWDY 2.000 3523 293 RTLVLLLLVL 2.000 3524 303 LTVFLLLVFY 2.000 3525 170 RCNLFRCRRL 2.000 3526 37 DVVLDDENPL 1.500 3527 31 VIWNTEDVVL 1.500 3528 298 LLLVLLTVFL 1.000 3529 317 QISQVIFRPL 1.000 3530 294 TLVLLLLVLL 1.000 3531 286 FIWRRYWRTL 1.000 3532 299 LLVLLTVFLL 1.000 3533 300 LVLLTVFLLL 1.000 3534 277 VNPLKTFVFF 1.000 3535 105 RSGPFALEEA 1.000 3536 302 LLTVFLLLVF 1.000 3537 74 FNSLTGEGNF 1.000 3538 231 ILTGKVEAEF 1.000 3539 80 EGNFNWRFVF 1.000 3540 297 LLLLVLLTVF 1.000 3541 165 NGAGPRCNLF 1.000 3542 276 FVNPLKTFVF 1.000 3543 113 EAEFRQPAVL 0.900 3544 185 VVKLKEAEDV 0.900 3545 214 KGRPEDLEFT 0.900 3546 162 LARNGAGPRC 0.900 3547 75 NSLTGEGNFN 0.750 3548 266 PSRPKTSFNW 0.750 3549 123 VLQVWDYDRI 0.600 3550 154 GPELCSVQLA 0.600 3551 84 NWRFVFRFDY 0.600 3552 211 RRRKGRPEDL 0.600 3553 61 KGLEHDKQET 0.600 3554 168 GPRCNLFRCR 0.600 3555 158 CSVQLARNGA 0.500 3556 283 FVFFIWRRYW 0.500 3557 76 SLTGEGNFNW 0.500 3558 9 DVPAPPPVDI 0.400 3559 261 EPLEKPSRPK 0.400 3560 29 RVVIWNTEDV 0.400 3561 21 RQPISYELRV 0.400 3562 251 RPVGKGRKQP 0.400 3563 309 LVFYTIPGQI 0.400 3564 6 FPQDVPAPPP 0.400 3565 258 KQPEPLEKPS 0.400 3566 117 RQPAVLVLQV 0.400 3567 313 TIPGQISQVI 0.400 3568 221 EFTDMGGNVY 0.400 3569 213 RKGRPEDLEF 0.300 3570 125 QVWDYDRISA 0.300 3571 129 YDRISANDFL 0.300 3572 102 VWRRSGPFAL 0.300 3573 115 EFRQPAVLVL 0.300 3574 288 WRRYWRTLVL 0.300 3575 134 ANDFLGSLEL 0.300 3576 78 TGEGNFNWRF 0.300 3577 38 VVLDDENPLT 0.300 3578 234 GKVEAEFELL 0.300 3579 65 HDKQETDVHF 0.300 3580 291 YWRTLVLLLL 0.300 3581 12 APPPVDIKPR 0.300 3582 131 RISANDFLGS 0.300 3583 44 NPLTGEMSSD 0.300 3584 51 SSDIYVKSWV 0.300 3585 290 RYWRTLVLLL 0.200 3586 282 TFVFFIWRRY 0.200 3587 183 WPVVKLKEAE 0.200 3588 10 VPAPPPVDIK 0.200 3589 68 QETDVHFNSL 0.200 3590 227 GNVYILTGKV 0.200 3591 93 YLPTEREVSV 0.200 3592 30 VVIWNTEDVV 0.200 3593 296 VLLLLVLLTV 0.200 3594 150 RGARGPELCS 0.200 3595 301 VLLTVFLLLV 0.200 3596 289 RRYWRTLVLL 0.200 3597 312 YTIPGQISQV 0.200 3598 187 KLKEAEDVER 0.180 3599 158P3D2 v.2a B35-10mers Pos 1234567890 Score SEQ. ID NO. 114 RPIKLLVRVY 80.000 3600 3 DPGDSDGVNL 60.000 3601 45 RSLEEEFNHF 30.000 3602 164 NPIFGEILEL 30.000 3603 110 IPQNRPIKLL 20.000 3604 215 RANCGLASQY 12.000 3605 177 LPAETELTVA 6.000 3606 211 YSHHRANCGL 5.000 3607 31 VSPKKAVATL 5.000 3608 134 DPNGKADPYV 4.000 3609 6 DSDGVNLISM 3.000 3610 121 RVYVVKATNL 2.000 3611 83 VGKFKGSFLI 1.200 3612 97 EAVLFSEPQI 1.200 3613 149 RERQDTKERY 1.200 3614 201 ETHIDLENRF 1.000 3615 91 LIYPESEAVL 1.000 3616 81 HLVGKFKGSF 1.000 3617 158 YIPKQLNPIF 1.000 3618 77 EGSGHLVGKF 1.000 3619 227 WVQQGPQEPF 1.000 3620 109 GIPQNRPIKL 1.000 3621 162 QLNPIFGEIL 1.000 3622 197 DLIGETHIDL 1.000 3623 183 LTVAVFEHDL 1.000 3624 82 LVGKFKGSFL 1.000 3625 174 SISLPAETEL 1.000 3626 38 ATLKIYNRSL 1.000 3627 161 KQLNPIFGEI 0.800 3628 145 VSAGRERQDT 0.750 3629 175 ISLPAETELT 0.750 3630 32 SPKKAVATLK 0.600 3631 93 YPESEAVLFS 0.600 3632 202 THIDLENRFY 0.600 3633 159 IPKQLNPIFG 0.600 3634 35 KAVATLKIYN 0.600 3635 73 GGEEEGSGHL 0.600 3636 101 FSEPQISRGI 0.600 3637 136 NGKADPYVVV 0.600 3638 218 CGLASQYEVW 0.500 3639 43 YNRSLEEEFN 0.450 3640 131 APADPNGKAD 0.400 3641 18 EIQDQGEAEV 0.400 3642 28 KGTVSPKKAV 0.400 3643 10 VNLISMVGEI 0.400 3644 34 KKAVATLKIY 0.400 3645 92 IYPESEAVLF 0.300 3646 130 LAPADPNGKA 0.300 3647 37 VATLKIYNRS 0.300 3648 186 AVFEHDLVGS 0.300 3649 124 VVKATNLAPA 0.300 3650 51 FNHFEDWLNV 0.300 3651 90 FLIYPESEAV 0.300 3652 184 TVAVFEHDLV 0.300 3653 207 ENRFYSHHRA 0.300 3654 21 DQGEAEVKGT 0.300 3655 119 LVRVYVVKAT 0.300 3656 179 AETELTVAVF 0.200 3657 61 FPLYRGQGGQ 0.200 3658 176 SLPAETELTV 0.200 3659 193 VGSDDLIGET 0.200 3660 217 NCGLASQYEV 0.200 3661 52 NHFEDWLNVF 0.200 3662 133 ADPNGKADPY 0.200 3663 219 GLASQYEVWV 0.200 3664 140 DPYVVVSAGR 0.200 3665 87 KGSFLIYPES 0.200 3666 55 EDWLNVFPLY 0.200 3667 84 GKFKGSFLIY 0.200 3668 103 EPQISRGIPQ 0.200 3669 138 KADPYVVVSA 0.180 3670 106 ISRGIPQNRP 0.150 3671 98 AVLFSEPQIS 0.150 3672 29 GTVSPKKAVA 0.100 3673 49 EEFNHFEDWL 0.100 3674 118 LLVRVYVVKA 0.100 3675 127 ATNLAPADPN 0.100 3676 172 ELSISLPAET 0.100 3677 42 IYNRSLEEEF 0.100 3678 30 TVSPKKAVAT 0.100 3679 168 GEILELSISL 0.100 3680 166 IFGEILELSI 0.080 3681 157 RYIPKQLNPI 0.080 3682 150 ERQDTKERYI 0.080 3683 13 ISMVGEIQDQ 0.075 3684 115 PIKLLVRVYV 0.060 3685 155 KERYIPKQLN 0.060 3686 153 DTKERYIPKQ 0.060 3687 126 KATNLAPADP 0.060 3688 147 AGRERQDTKE 0.060 3689 22 QGEAEVKGTV 0.060 3690 173 LSISLPAETE 0.050 3691 78 GSGHLVGKFK 0.050 3692 88 GSFLIYPESE 0.050 3693 221 ASQYEVWVQQ 0.050 3694 220 LASQYEVWVQ 0.045 3695 167 FGEILELSIS 0.045 3696 16 VGEIQDQGEA 0.045 3697 107 SRGIPQNRPI 0.040 3698 190 HDLVGSDDLI 0.040 3699 158P3D2 v.3 B35-10mers Pos 1234567890 Score SEQ. ID NO. 7 VSVRRRSGPF 5.000 3700 1 LPTEREVSVR 0.600 3701 8 SVRRRSGPFA 0.300 3702 9 VRRRSGPFAL 0.300 3703 6 EVSVRRRSGP 0.010 3704 10 RRRSGPFALE 0.006 3705 3 TEREVSVRRR 0.006 3706 4 EREVSVRRRS 0.003 3707 5 REVSVRRRSG 0.002 3708 2 PTEREVSVRR 0.000 3709 158P3D2 v.4 B35-10mers Pos 1234567890 Score SEQ. ID NO. 2 LPTEREVSIW 30.000 3710 8 VSIWRRSGPF 5.000 3711 1 YLPTEREVSI 0.400 3712 10 IWRRSGPFAL 0.300 3713 9 SIWRRSGPFA 0.100 3714 7 EVSIWRRSGP 0.010 3715 4 TEREVSIWRR 0.006 3716 5 EREVSIWRRS 0.003 3717 6 REVSIWRRSG 0.002 3718 3 PTEREVSIWR 0.000 3719 158P3D2 v.5a B35-10mers Pos 1234567890 Score SEQ. ID NO. 16 TSLDPWSCSY 20.000 3720 19 DPWSCSYQTW 10.000 3721 36 SSALCSWPAM 10.000 3722 31 GPGAPSSALC 2.000 3723 42 WPAMGPGRGA 2.000 3724 46 GPGRGAICFA 2.000 3725 13 LPMTSLDPWS 2.000 3726 33 GAPSSALCSW 1.500 3727 30 VGPGAPSSAL 1.000 3728 21 WSCSYQTWCV 1.000 3729 9 YTASLPMTSL 1.000 3730 45 MGPGRGAICF 1.000 3731 4 LQVWDYTASL 1.000 3732 12 SLPMTSLDPW 0.500 3733 49 RGAICFAAAA 0.200 3734 34 APSSALCSWP 0.200 3735 3 VLQVWDYTAS 0.150 3736 43 PAMGPGRGAI 0.120 3737 15 MTSLDPWSCS 0.100 3738 1 VLVLQVWDYT 0.100 3739 44 AMGPGRGAIC 0.100 3740 25 YQTWCVGPGA 0.100 3741 2 LVLQVWDYTA 0.100 3742 29 CVGPGAPSSA 0.100 3743 28 WCVGPGAPSS 0.100 3744 6 VWDYTASLPM 0.060 3745 40 CSWPAMGPGR 0.050 3746 35 PSSALCSWPA 0.050 3747 11 ASLPMTSLDP 0.050 3748 23 CSYQTWCVGP 0.050 3749 47 PGRGAICFAA 0.030 3750 37 SALCSWPAMG 0.030 3751 10 TASLPMTSLD 0.030 3752 5 QVWDYTASLP 0.020 3753 14 PMTSLDPWSC 0.015 3754 27 TWCVGPGAPS 0.010 3755 26 QTWCVGPGAP 0.010 3756 7 WDYTASLPMT 0.010 3757 48 GRGAICFAAA 0.010 3758 22 SCSYQTWCVG 0.010 3759 39 LCSWPAMGPG 0.010 3760 38 ALCSWPAMGP 0.010 3761 18 LDPWSCSYQT 0.010 3762 8 DYTASLPMTS 0.010 3763 32 PGAPSSALCS 0.010 3764 17 SLDPWSCSYQ 0.003 3765 24 SYQTWCVGPG 0.001 3766 20 PWSCSYQTWC 0.001 3767 41 SWPAMGPGRG 0.001 3768

TABLE XIXA MHC Class I Analysis of 158P3D2 (9-mers) part 1: MHC Class I nonamer analysis of 58P3D2 v.1 (aa 1-328) Listed are scores which correlate with the ligation strength to a defined HLA type for a sequence of amino acids. The algorithms used are based on the book “MHC Ligands and Peptide Motifs” by H. G. Rammensee, J. Bachmann and S. Stevanovic. The probability of being processed and presented is given in order to predict T-cell epitopes HLA-A*0201 nonamers Pos 1 2 3 4 5 6 7 8 9 score Seq. ID. NO 297 L L L L V L L T V 31 3769 299 L L V L L T V F L 27 3770 302 L L T V F L L L V 27 3771 294 T L V L L L L V L 26 3772 133 S A N D F L G S L 25 3773 295 L V L L L L V L L 25 3774 224 D M G G N V Y I L 24 3775 301 V L L T V F L L L 24 3776 306 F L L L V F Y T I 24 3777 313 T I P G Q I S Q V 24 3778 137 F L G S L E L Q L 23 3779 178 R L R G W W P V V 23 3780 296 V L L L L V L L T 23 3781 230 Y I L T G K V E A 22 3782 293 R T L V L L L L V 22 3783 300 L V L L T V F L L 22 3784  31 V I W N T E D V V 20 3785 140 S L E L Q L P D M 20 3786 144 Q L P D M V R G A 20 3787 152 A R G P E L C S V 20 3788 180 R G W W P V V K L 20 3789 228 N V Y I L T G K V 20 3790   2 W I D I F P Q D V 19 3791  30 V V I W N T E D V 19 3792  38 V V L D D E N P L 19 3793  55 Y V K S W V K G L 19 3794 231 I L T G K V E A E 19 3795 272 S F N W F V N P L 19 3796 276 F V N P L K T F V 19 3797 279 P L K T F V F F I 19 3798 298 L L L V L L T V F 19 3799  23 P I S Y E L R V V 18 3800 116 F R Q P A V L V L 18 3801 118 Q P A V L V L Q V 18 3802 291 Y W R T L V L L L 18 3803  39 V L D D E N P L T 17 3804  94 L P T E R E V S V 17 3805 290 R Y W R T L V L L 17 3806   4 D I F P Q D V P A 16 3807  10 V P A P P P V D I 16 3808  24 I S Y E L R V V I 16 3809  46 L T G E M S S D I 16 3810  62 G L E H D K Q E T 16 3811 135 N D F L G S L E L 16 3812 237 E A E F E L L T V 16 3813  27 E L R V V I W N T 15 3814  32 I W N T E D V V L 15 3815  92 D Y L P T E R E V 15 3816 114 A E F R Q P A V L 15 3817 121 V L V L Q V W D Y 15 3818 141 L E L Q L P D M V 15 3819 161 Q L A R N G A G P 15 3820 165 N G A G P R C N L 15 3821 223 T D M G G N V Y I 15 3822 234 G K V E A E F E L 15 3823 242 L L T V E E A E K 15 3824 287 I W R R Y W R T L 15 3825 307 L L L V F Y T I P 15 3826 HLA-A1 nonamers Pos 1 2 3 4 5 6 7 8 9 score Seq. ID. NO 222 F T D M G G N V Y 36 3827  34 N T E D V V L D D 25 3828  47 T G E M S S D I Y 25 3829  18 I K P R Q P I S Y 21 3830 121 V L V L Q V W D Y 20 3831  69 E T D V H F N S L 19 3832  51 S S D I Y V K S W 18 3833  95 P T E R E V S V W 18 3834 312 Y T I P G Q I S Q 18 3835 HLA-A26 nonamers Pos 1 2 3 4 5 6 7 8 9 score Seq. ID. NO  69 E T D V H F N S L 30 3836 304 T V F L L L V F Y 28 3837  55 Y V K S W V K G L 25 3838 303 L T V F L L L V F 25 3839 295 L V L L L L V L L 24 3840 121 V L V L Q V W D Y 23 3841 232 L T G K V E A E F 23 3842 283 F V F F I W R R Y 23 3843 298 L L L V L L T V F 23 3844   4 D I F P Q D V P A 22 3845 140 S L E L Q L P D M 22 3846 235 K V E A E F E L L 22 3847 300 L V L L T V F L L 22 3848 222 F T D M G G N V Y 21 3849 294 T L V L L L L V L 21 3850  17 D I K P R Q P I S 20 3851  66 D K Q E T D V H F 20 3852 101 S V W R R S G P F 20 3853 224 D M G G N V Y I L 20 3854 275 W F V N P L K T F 20 3855 301 V L L T V F L L L 20 3856 313 T I P G Q I S Q V 20 3857  27 E L R V V I W N T 19 3858  38 V V L D D E N P L 19 3859 108 P F A L E E A E F 19 3860 136 D F L G S L E L Q 19 3861 137 F L G S L E L Q L 19 3862   9 D V P A P P P V D 18 3863  42 D E N P L T G E M 18 3864  86 R F V F R F D Y L 18 3865 193 D V E R E A Q E A 18 3866 272 S F N W F V N P L 18 3867 299 L L V L L T V F L 18 3868 309 L V F Y T I P G Q 18 3869  37 D V V L D D E N P 17 3870  53 D I Y V K S W V K 17 3871  99 E V S V W R R S G 17 3872 130 D R I S A N D F L 17 3873  45 P L T G E M S S D 16 3874  71 D V H F N S L T G 16 3875 156 E L C S V Q L A R 16 3876 219 D L E F T D M G G 16 3877 231 I L T G K V E A E 16 3878 268 R P K T S F N W F 16 3879 278 N P L K T F V F F 16 3880 281 K T F V F F I W R 16 3881 317 Q I S Q V I F R P 16 3882  34 N T E D V V L D D 15 3883  83 F N W R F V F R F 15 3884  95 P T E R E V S V W 15 3885 142 E L Q L P D M V R 15 3886 144 Q L P D M V R G A 15 3887 239 E F E L L T V E E 15 3888 241 E L L T V E E A E 15 3889 286 F I W R R Y W R T 15 3890 293 R T L V L L L L V 15 3891 312 Y T I P G Q I S Q 15 3892 HLA-A3 nonamers Pos 1 2 3 4 5 6 7 8 9 score Seq. ID. NO 320 Q V I F R P L H K 31 3893  53 D I Y V K S W V K 27 3894  59 W V K G L E H D K 23 3895 178 R L R G W W P V V 23 3896 242 L L T V E E A E K 23 3897 161 Q L A R N G A G P 22 3898 101 S V W R R S G P F 21 3899 257 R K Q P E P L E K 21 3900 297 L L L L V L L T V 21 3901 298 L L L V L L T V F 21 3902 304 T V F L L L V F Y 21 3903 120 A V L V L Q V W D 20 3904 142 E L Q L P D M V R 20 3905 262 P L E K P S R P K 20 3906 156 E L C S V Q L A R 19 3907 179 L R G W W P V V K 19 3908 187 K L K E A E D V E 19 3909 198 A Q E A Q A G K K 19 3910 172 N L F R C R R L R 18 3911 294 T L V L L L L V L 18 3912 306 F L L L V F Y T I 18 3913   9 D V P A P P P V D 17 3914  45 P L T G E M S S D 17 3915  71 D V H F N S L T G 17 3916 121 V L V L Q V W D Y 17 3917 148 M V R G A R G P E 17 3918 201 A Q A G K K K R K 17 3919 206 K K R K Q R R R K 17 3920 247 E A E K R P V G K 17 3921 289 R R Y W R T L V L 17 3922 295 L V L L L L V L L 17 3923  15 P V D I K P R Q P 16 3924  24 I S Y E L R V V I 16 3925  29 R V V I W N T E D 16 3926  76 S L T G E G N F N 16 3927 137 F L G S L E L Q L 16 3928 185 V V K L K E A E D 16 3929 193 D V E R E A Q E A 16 3930 199 Q E A Q A G K K K 16 3931 214 K G R P E D L E F 16 3932 228 N V Y I L T G K V 16 3933 230 Y I L T G K V E A 16 3934 231 I L T G K V E A E 16 3935 250 K R P V G K G R K 16 3936 252 P V G K G R K Q P 16 3937 283 F V F F I W R R Y 16 3938 296 V L L L L V L L T 16 3939 301 V L L T V F L L L 16 3940 313 T I P G Q I S Q V 16 3941   4 D I F P Q D V P A 15 3942  93 Y L P T E R E V S 15 3943 222 F T D M G G N V Y 15 3944 235 K V E A E F E L L 15 3945 299 L L V L L T V F L 15 3946 HLA-B*0702 nonamers Pos 1 2 3 4 5 6 7 8 9 score Seq. ID. NO  10 V P A P P P V D I 23 3947 265 K P S R P K T S F 23 3948 154 G P E L C S V Q L 22 3949 278 N P L K T F V F F 21 3950 118 Q P A V L V L Q V 20 3951 314 I P G Q I S Q V I 19 3952  22 Q P I S Y E L R V 18 3953  94 L P T E R E V S V 18 3954 268 R P K T S F N W F 18 3955 165 N G A G P R C N L 17 3956 180 R G W W P V V K L 17 3957  19 K P R Q P I S Y E 16 3958 183 W P V V K L K E A 16 3959 116 F R Q P A V L V L 15 3960 255 K G R K Q P E P L 15 3961 289 R R Y W R T L V L 15 3962 291 Y W R T L V L L L 15 3963  32 I W N T E D V V L 14 3964 114 A E F R Q P A V L 14 3965 115 E F R Q P A V L V 14 3966 149 V R G A R G P E L 14 3967 224 D M G G N V Y I L 14 3968 251 R P V G K G R K Q 14 3969 299 L L V L L T V F L 14 3970  12 A P P P V D I K P 13 3971  69 E T D V H F N S L 13 3972 103 W R R S G P F A L 13 3973 137 F L G S L E L Q L 13 3974 145 L P D M V R G A R 13 3975 178 R L R G W W P V V 13 3976 212 R R K G R P E D L 13 3977 235 K V E A E F E L L 13 3978 287 I W R R Y W R T L 13 3979 290 R Y W R T L V L L 13 3980 294 T L V L L L L V L 13 3981 301 V L L T V F L L L 13 3982 318 I S Q V I F R P L 13 3983   6 F P Q D V P A P P 12 3984  16 V D I K P R Q P I 12 3985  86 R F V F R F D Y L 12 3986 107 G P F A L E E A E 12 3987 135 N D F L G S L E L 12 3988 168 G P R C N L F R C 12 3989 214 K G R P E D L E F 12 3990 259 Q P E P L E K P S 12 3991 272 S F N W F V N P L 12 3992 292 W R T L V L L L L 12 3993 295 L V L L L L V L L 12 3994  13 P P P V D I K P R 11 3995  14 P P V D I K P R Q 11 3996  20 P R Q P I S Y E L 11 3997  24 I S Y E L R V V I 11 3998  38 V V L D D E N P L 11 3999  55 Y V K S W V K G L 11 4000  88 V F R F D Y L P T 11 4001 102 V W R R S G P F A 11 4002 130 D R I S A N D F L 11 4003 216 R P E D L E F T D 11 4004 223 T D M G G N V Y I 11 4005 261 E P L E K P S R P 11 4006 300 L V L L T V F L L 11 4007 HLA-B*08 nonamers Pos 1 2 3 4 5 6 7 8 9 score Seq. ID. NO 212 R R K G R P E D L 28 4008 185 V V K L K E A E D 23 4009 279 P L K T F V F F I 23 4010  17 D I K P R Q P I S 22 4011  55 Y V K S W V K G L 22 4012 268 R P K T S F N W F 22 4013 203 A G K K K R K Q R 21 4014 149 V R G A R G P E L 20 4015 205 K K K R K Q R R R 20 4016 261 E P L E K P S R P 20 4017 154 G P E L C S V Q L 19 4018 166 G A G P R C N L F 19 4019 183 W P V V K L K E A 19 4020 204 G K K K R K Q R R 19 4021 231 I L T G K V E A E 19 4022 253 V G K G R K Q P E 19 4023  86 R F V F R F D Y L 18 4024 171 C N L F R C R R L 18 4025 187 K L K E A E D V E 18 4026 207 K R K Q R R R K G 18 4027 277 V N P L K T F V F 18 4028 289 R R Y W R T L V L 18 4029 299 L L V L L T V F L 18 4030  94 L P T E R E V S V 17 4031 103 W R R S G P F A L 17 4032 137 F L G S L E L Q L 17 4033 287 I W R R Y W R T L 17 4034 291 Y W R T L V L L L 17 4035 294 T L V L L L L V L 17 4036 301 V L L T V F L L L 17 4037  27 E L R V V I W N T 16 4038 101 S V W R R S G P F 16 4039 133 S A N D F L G S L 16 4040 210 Q R R R K G R P E 16 4041 251 R P V G K G R K Q 16 4042 255 K G R K Q P E P L 16 4043 266 P S R P K T S F N 16 4044  53 D I Y V K S W V K 15 4045 113 E A E F R Q P A V 15 4046 176 C R R L R G W W P 15 4047 247 E A E K R P V G K 15 4048  10 V P A P P P V D I 14 4049 173 L F R C R R L R G 14 4050 202 Q A G K K K R K Q 14 4051 209 K Q R R R K G R P 14 4052 234 G K V E A E F E L 14 4053 246 E E A E K R P V G 14 4054 306 F L L L V F Y T I 14 4055 HLA-B*1510 nonamers Pos 1 2 3 4 5 6 7 8 9 score Seq. ID. NO  32 I W N T E D V V L 16 4056 116 F R Q P A V L V L 15 4057 287 I W R R Y W R T L 15 4058 318 I S Q V I F R P L 15 4059 154 G P E L C S V Q L 14 4060 165 N G A G P R C N L 14 4061 171 C N L F R C R R L 14 4062 180 R G W W P V V K L 14 4063  20 P R Q P I S Y E L 13 4064 103 W R R S G P F A L 13 4065 114 A E F R Q P A V L 13 4066 224 D M G G N V Y I L 13 4067 234 G K V E A E F E L 13 4068 294 T L V L L L L V L 13 4069  55 Y V K S W V K G L 12 4070  64 E H D K Q E T D V 12 4071  69 E T D V H F N S L 12 4072 135 N D F L G S L E L 12 4073 149 V R G A R G P E L 12 4074 212 R R K G R P E D L 12 4075 255 K G R K Q P E P L 12 4076 289 R R Y W R T L V L 12 4077 290 R Y W R T L V L L 12 4078 291 Y W R T L V L L L 12 4079 295 L V L L L L V L L 12 4080 299 L L V L L T V F L 12 4081  38 V V L D D E N P L 11 4082 133 S A N D F L G S L 11 4083 235 K V E A E F E L L 11 4084 272 S F N W F V N P L 11 4085 300 L V L L T V F L L 11 4086  72 V H F N S L T G E 10 4087  81 G N F N W R F V F 10 4088  86 R F V F R F D Y L 10 4089 130 D R I S A N D F L 10 4090 137 F L G S L E L Q L 10 4091 292 W R T L V L L L L 10 4092 301 V L L T V F L L L 10 4093  42 D E N P L T G E M 9 4094  66 D K Q E T D V H F 9 4095  79 G E G N F N W R F 9 4096  83 F N W R F V F R F 9 4097 166 G A G P R C N L F 9 4098 214 K G R P E D L E F 9 4099 265 K P S R P K T S F 9 4100 278 N P L K T F V F F 9 4101 298 L L L V L L T V F 9 4102  24 I S Y E L R V V I 8 4103 108 P F A L E E A E F 8 4104 140 S L E L Q L P D M 8 4105 232 L T G K V E A E F 8 4106 275 W F V N P L K T F 8 4107 277 V N P L K T F V F 8 4108 303 L T V F L L L V F 8 4109 315 P G Q I S Q V I F 8 4110 HLA-B*2705 nonamers Pos 1 2 3 4 5 6 7 8 9 score Seq. ID. NO 289 R R Y W R T L V L 28 4111 250 K R P V G K G R K 27 4112 212 R R K G R P E D L 26 4113  20 P R Q P I S Y E L 25 4114  97 E R E V S V W R R 25 4115 116 F R Q P A V L V L 24 4116 292 W R T L V L L L L 24 4117 130 D R I S A N D F L 23 4118 103 W R R S G P F A L 22 4119 149 V R G A R G P E L 22 4120 179 L R G W W P V V K 22 4121  85 W R F V F R F D Y 21 4122 169 P R C N L F R C R 21 4123 211 R R R K G R P E D 20 4124 135 N D F L G S L E L 19 4125 177 R R L R G W W P V 19 4126 180 R G W W P V V K L 19 4127  90 R F D Y L P T E R 18 4128 104 R R S G P F A L E 18 4129 204 G K K K R K Q R R 18 4130 227 G N V Y I L T G K 18 4131 257 R K Q P E P L E K 18 4132  79 G E G N F N W R F 17 4133  81 G N F N W R F V F 17 4134 154 G P E L C S V Q L 17 4135 170 R C N L F R C R R 17 4136 201 A Q A G K K K R K 17 4137 205 K K K R K Q R R R 17 4138 214 K G R P E D L E F 17 4139 256 G R K Q P E P L E 17 4140 265 K P S R P K T S F 17 4141 282 T F V F F I W R R 17 4142 298 L L L V L L T V F 17 4143 316 G Q I S Q V I F R 17 4144  53 D I Y V K S W V K 16 4145  75 N S L T G E G N F 16 4146  89 F R F D Y L P T E 16 4147 114 A E F R Q P A V L 16 4148 163 A R N G A G P R C 16 4149 181 G W W P V V K L K 16 4150 206 K K R K Q R R R K 16 4151 207 K R K Q R R R K G 16 4152 234 G K V E A E F E L 16 4153 243 L T V E E A E K R 16 4154 268 R P K T S F N W F 16 4155 281 K T F V F F I W R 16 4156 290 R Y W R T L V L L 16 4157 294 T L V L L L L V L 16 4158 295 L V L L L L V L L 16 4159  21 R Q P I S Y E L R 15 4160  32 I W N T E D V V L 15 4161  49 E M S S D I Y V K 15 4162  86 R F V F R F D Y L 15 4163 109 F A L E E A E F R 15 4164 142 E L Q L P D M V R 15 4165 152 A R G P E L C S V 15 4166 165 N G A G P R C N L 15 4167 166 G A G P R C N L F 15 4168 200 E A Q A G K K K R 15 4169 203 A G K K K R K Q R 15 4170 215 G R P E D L E F T 15 4171 232 L T G K V E A E F 15 4172 260 P E P L E K P S R 15 4173 267 S R P K T S F N W 15 4174 278 N P L K T F V F F 15 4175 303 L T V F L L L V F 15 4176 304 T V F L L L V F Y 15 4177  28 L R V V I W N T E 14 4178  96 T E R E V S V W R 14 4179 108 P F A L E E A E F 14 4180 156 E L C S V Q L A R 14 4181 171 C N L F R C R R L 14 4182 188 L K E A E D V E R 14 4183 197 E A Q E A Q A G K 14 4184 198 A Q E A Q A G K K 14 4185 199 Q E A Q A G K K K 14 4186 208 R K Q R R R K G R 14 4187 224 D M G G N V Y I L 14 4188 255 K G R K Q P E P L 14 4189 262 P L E K P S R P K 14 4190 275 W F V N P L K T F 14 4191 283 F V F F I W R R Y 14 4192 299 L L V L L T V F L 14 4193 300 L V L L T V F L L 14 4194 301 V L L T V F L L L 14 4195 315 P G Q I S Q V I F 14 4196 HLA-B*2709 nonamers Pos 1 2 3 4 5 6 7 8 9 score Seq. ID. NO 289 R R Y W R T L V L 27 4197 177 R R L R G W W P V 24 4198 212 R R K G R P E D L 24 4199  20 P R Q P I S Y E L 23 4200 116 F R Q P A V L V L 23 4201 130 D R I S A N D F L 22 4202 292 W R T L V L L L L 22 4203 103 W R R S G P F A L 21 4204 149 V R G A R G P E L 21 4205 152 A R G P E L C S V 20 4206 288 W R R Y W R T L V 18 4207 180 R G W W P V V K L 16 4208  86 R F V F R F D Y L 15 4209 154 G P E L C S V Q L 15 4210 211 R R R K G R P E D 15 4211 290 R Y W R T L V L L 15 4212 293 R T L V L L L L V 15 4213 104 R R S G P F A L E 14 4214 215 G R P E D L E F T 14 4215 234 G K V E A E F E L 14 4216 256 G R K Q P E P L E 14 4217  38 V V L D D E N P L 13 4218  81 G N F N W R F V F 13 4219 89 F R F D Y L P T E 13 4220 114 A E F R Q P A V L 13 4221 135 N D F L G S L E L 13 4222 137 F L G S L E L Q L 13 4223 163 A R N G A G P R C 13 4224 171 C N L F R C R R L 13 4225 178 R L R G W W P V V 13 4226 250 K R P V G K G R K 13 4227 295 L V L L L L V L L 13 4228 300 L V L L T V F L L 13 4229 301 V L L T V F L L L 13 4230 HLA-B*4402 nonamers Pos 1 2 3 4 5 6 7 8 9 score Seq. ID. NO 114 A E F R Q P A V L 28 4231  79 G E G N F N W R F 21 4232 191 A E D V E R E A Q 17 4233 238 A E F E L L T V E 17 4234 116 F R Q P A V L V L 16 4235 166 G A G P R C N L F 16 4236 248 A E K R P V G K G 16 4237  25 S Y E L R V V I W 15 4238  51 S S D I Y V K S W 15 4239  69 E T D V H F N S L 15 4240  81 G N F N W R F V F 15 4241 135 N D F L G S L E L 15 4242 214 K G R P E D L E F 15 4243 263 L E K P S R P K T 15 4244 275 W F V N P L K T F 15 4245 295 L V L L L L V L L 15 4246 301 V L L T V F L L L 15 4247 304 T V F L L L V F Y 15 4248  18 I K P R Q P I S Y 14 4249  20 P R Q P I S Y E L 14 4250  26 Y E L R V V I W N 14 4251  42 D E N P L T G E M 14 4252 174 F R C R R L R G W 14 4253 246 E E A E K R P V G 14 4254 277 V N P L K T F V F 14 4255 278 N P L K T F V F F 14 4256 283 F V F F I W R R Y 14 4257 284 V F F I W R R Y W 14 4258 289 R R Y W R T L V L 14 4259 290 R Y W R T L V L L 14 4260 291 Y W R T L V L L L 14 4261 292 W R T L V L L L L 14 4262 294 T L V L L L L V L 14 4263 300 L V L L T V F L L 14 4264 HLA-B*5101 nonamers Pos 1 2 3 4 5 6 7 8 9 score Seq. ID. NO 314 I P G Q I S Q V I 25 4265  10 V P A P P P V D I 23 4266  94 L P T E R E V S V 23 4267  24 I S Y E L R V V I 22 4268 237 E A E F E L L T V 22 4269  22 Q P I S Y E L R V 21 4270 118 Q P A V L V L Q V 21 4271 113 E A E F R Q P A V 18 4272 133 S A N D F L G S L 18 4273 180 R G W W P V V K L 18 4274 297 L L L L V L L T V 18 4275 306 F L L L V F Y T I 18 4276 154 G P E L C S V Q L 17 4277 261 E P L E K P S R P 17 4278 278 N P L K T F V F F 17 4279 310 V F Y T I P G Q I 17 4280  12 A P P P V D I K P 16 4281  92 D Y L P T E R E V 16 4282 109 F A L E E A E F R 16 4283 119 P A V L V L Q V W 16 4284   6 F P Q D V P A P P 15 4285  46 L T G E M S S D I 15 4286  80 E G N F N W R F V 15 4287 202 Q A G K K K R K Q 15 4288 228 N V Y I L T G K V 15 4289 251 R P V G K G R K Q 15 4290  31 V I W N T E D V V 14 4291  44 N P L T G E M S S 14 4292 145 L P D M V R G A R 14 4293 165 N G A G P R C N L 14 4294 190 E A E D V E R E A 14 4295 200 E A Q A G K K K R 14 4296 223 T D M G G N V Y I 14 4297 255 K G R K Q P E P L 14 4298 268 R P K T S F N W F 14 4299 289 R R Y W R T L V L 14 4300 301 V L L T V F L L L 14 4301  11 P A P P P V D I K 13 4302  13 P P P V D I K P R 13 4303  23 P I S Y E L R V V 13 4304  32 I W N T E D V V L 13 4305 116 F R Q P A V L V L 13 4306 124 L Q V W D Y D R I 13 4307 141 L E L Q L P D M V 13 4308 162 L A R N G A G P R 13 4309 183 W P V V K L K E A 13 4310 186 V K L K E A E D V 13 4311 216 R P E D L E F T D 13 4312 224 D M G G N V Y I L 13 4313 247 E A E K R P V G K 13 4314 279 P L K T F V F F I 13 4315 300 L V L L T V F L L 13 4316  14 P P V D I K P R Q 12 4317  16 V D I K P R Q P I 12 4318  53 D I Y V K S W V K 12 4319 107 G P F A L E E A E 12 4320 151 G A R G P E L C S 12 4321 153 R G P E L C S V Q 12 4322 168 G P R C N L F R C 12 4323 178 R L R G W W P V V 12 4324 197 E A Q E A Q A G K 12 4325 287 I W R R Y W R T L 12 4326 291 Y W R T L V L L L 12 4327 293 R T L V L L L L V 12 4328 294 T L V L L L L V L 12 4329 295 L V L L L L V L L 12 4330 302 L L T V F L L L V 12 4331 313 T I P G Q I S Q V 12 4332 part 2: MHC Class I nonamer analysis of 158P3D2 v.2a (aa 1-236). HLA-A*0201 nonamers Pos 1 2 3 4 5 6 7 8 9 score Seq. ID. NO 117 K L L V R V Y V V 28 4333  11 N L I S M V G E I 26 4334 165 P I F G E I L E L 25 4335  91 L I Y P E S E A V 24 4336 158 Y I P K Q L N P I 23 4337 162 Q L N P I F G E I 23 4338  39 T L K I Y N R S L 22 4339 169 E I L E L S I S L 22 4340 198 L I G E T H I D L 22 4341  32 S P K K A V A T L 19 4342  90 F L I Y P E S E A 19 4343 114 R P I K L L V R V 19 4344 119 L V R V Y V V K A 19 4345 177 L P A E T E L T V 19 4346 179 A E T E L T V A V 19 4347 191 D L V G S D D L I 19 4348 220 L A S Q Y E V W V 19 4349  19 I Q D Q G E A E V 18 4350 175 I S L P A E T E L 18 4351 176 S L P A E T E L T 18 4352 184 T V A V F E H D L 18 4353   5 G D S D G V N L I 17 4354  23 G E A E V K G T V 17 4355 137 G K A D P Y V V V 17 4356  14 S M V G E I Q D Q 16 4357  29 G T V S P K K A V 16 4358  34 K K A V A T L K I 16 4359  92 I Y P E S E A V L 16 4360  98 A V L F S E P Q I 16 4361 116 I K L L V R V Y V 16 4362 197 D L I G E T H I D 16 4363 228 E A E F E L L T V 16 4364 105 Q I S R G I P Q N 15 4365 129 N L A P A D P N G 15 4366 170 I L E L S I S L P 15 4367 215 R A N C G L A S Q 15 4368 218 C G L A S Q Y E V 15 4369  8 D G V N L I S M V 14 4370  46 S L E E E F N H F 14 4371  52 N H F E D W L N V 14 4372  74 G E E E G S G H L 14 4373 110 I P Q N R P I K L 14 4374 118 L L V R V Y V V K 14 4375 138 K A D P Y V V V S 14 4376 185 V A V F E H D L V 14 4377 190 H D L V G S D D L 14 4378 HLA-A1 nonamers Pos 1 2 3 4 5 6 7 8 9 score Seq. ID. NO 203 H I D L E N R F Y 26 4379  85 K F K G S F L I Y 25 4380  6 D S D G V N L I S 24 4381  56 D W L N V F P L Y 22 4382 101 F S E P Q I S R G 21 4383 180 E T E L T V A V F 18 4384 115 P I K L L V R V Y 17 4385 138 K A D P Y V V V S 17 4386 167 F G E I L E L S I 17 4387 216 A N C G L A S Q Y 17 4388  1 M D D P G D S D G 16 4389  46 S L E E E F N H F 16 4390  95 E S E A V L F S E 16 4391 134 D P N G K A D P Y 16 4392  35 K A V A T L K I Y 15 4393 132 P A D P N G K A D 15 4394 150 E R Q D T K E R Y 15 4395  69 G Q D G G G E E E 14 4396  75 E E E G S G H L V 14 4397  93 Y P E S E A V L F 14 4398 148 G R E R Q D T K E 14 4399 194 G S D D L I G E T 14 4400 205 D L E N R F Y S H 14 4401  16 V G E I Q D Q G E 13 4402 154 T K E R Y I P K Q 13 4403 170 I L E L S I S L P 13 4404 189 E H D L V G S D D 13 4405 195 S D D L I G E T H 13 4406 HLA-A26 nonamers Pos 1 2 3 4 5 6 7 8 9 score Seq. ID. NO 180 E T E L T V A V F 31 4407 169 E I L E L S I S L 28 4408 165 P I F G E I L E L 27 4409 115 P I K L L V R V Y 26 4410  46 S L E E E F N H F 25 4411  26 E V K G T V S P K 24 4412  85 K F K G S F L I Y 24 4413 153 D T K E R Y I P K 23 4414  82 L V G K F K G S F 22 4415  53 H F E D W L N V F 21 4416  56 D W L N V F P L Y 21 4417 172 E L S I S L P A E 21 4418 201 E T H I D L E N R 21 4419 203 H I D L E N R F Y 21 4420  50 E F N H F E D W L 20 4421  59 N V F P L Y R G Q 20 4422 198 L I G E T H I D L 20 4423  18 E I Q D Q G E A E 19 4424 134 D P N G K A D P Y 19 4425 182 E L T V A V F E H 19 4426 184 T V A V F E H D L 19 4427 205 D L E N R F Y S H 19 4428  39 T L K I Y N R S L 18 4429  55 E D W L N V F P L 18 4430 150 E R Q D T K E R Y 18 4431 197 D L I G E T H I D 18 4432 158 Y I P K Q L N P I 17 4433 191 D L V G S D D L I 17 4434 225 E V W V Q Q G P Q 17 4435  11 N L I S M V G E I 16 4436  38 A T L K I Y N R S 16 4437  78 G S G H L V G K F 16 4438  81 H L V G K F K G S 16 4439 105 Q I S R G I P Q N 16 4440 119 L V R V Y V V K A 16 4441  32 S P K K A V A T L 15 4442 162 Q L N P I F G E I 15 4443 183 L T V A V F E H D 15 4444 202 T H I D L E N R F 15 4445 216 A N C G L A S Q Y 15 4446 219 G L A S Q Y E V W 15 4447 HLA-A3 nonamers Pos 1 2 3 4 5 6 7 8 9 score Seq. ID. NO 118 L L V R V Y V V K 32 4448  26 E V K G T V S P K 26 4449 121 R V Y V V K A T N 26 4450 147 A G R E R Q D T K 23 4451  20 Q D Q G E A E V K 21 4452  30 T V S P K K A V A 21 4453  41 K I Y N R S L E E 21 4454 117 K L L V R V Y V V 21 4455 186 A V F E H D L V G 21 4456  62 P L Y R G Q G G Q 20 4457 115 P I K L L V R V Y 20 4458 216 A N C G L A S Q Y 20 4459 109 G I P Q N R P I K 19 4460 205 D L E N R F Y S H 19 4461  33 P K K A V A T L K 18 4462  57 W L N V F P L Y R 18 4463  98 A V L F S E P Q I 18 4464 105 Q I S R G I P Q N 18 4465  82 L V G K F K G S F 17 4466 119 L V R V Y V V K A 17 4467 124 V V K A T N L A P 17 4468 143 V V V S A G R E R 17 4469 174 S I S L P A E T E 17 4470  9 G V N L I S M V G 16 4471  46 S L E E E F N H F 16 4472  77 E G S G H L V G K 16 4473  79 S G H L V G K F K 16 4474  90 F L I Y P E S E A 16 4475  91 L I Y P E S E A V 16 4476 162 Q L N P I F G E I 16 4477 170 I L E L S I S L P 16 4478 HLA-B*0702 nonamers Pos 1 2 3 4 5 6 7 8 9 score Seq. ID. NO  32 S P K K A V A T L 23 4479 131 A P A D P N G K A 22 4480 110 I P Q N R P I K L 21 4481 114 R P I K L L V R V 20 4482 177 L P A E T E L T V 19 4483  93 Y P E S E A V L F 18 4484 159 I P K Q L N P I F 18 4485  4 P G D S D G V N L 14 4486 165 P I F G E I L E L 14 4487  55 E D W L N V F P L 13 4488  92 I Y P E S E A V L 13 4489 111 P Q N R P I K L L 13 4490 134 D P N G K A D P Y 13 4491 137 G K A D P Y V V V 13 4492 155 K E R Y I P K Q L 13 4493 175 I S L P A E T E L 13 4494  3 D P G D S D G V N 12 4495  83 V G K F K G S F L 12 4496 103 E P Q I S R G I P 12 4497 140 D P Y V V V S A G 12 4498 179 A E T E L T V A V 12 4499 228 V Q Q G P Q E P F 12 4500  30 T V S P K K A V A 11 4501  34 K K A V A T L K I 11 4502  50 E F N H F E D W L 11 4503  61 F P L Y R G Q G G 11 4504 112 Q N R P I K L L V 11 4505 119 L V R V Y V V K A 11 4506 122 V Y V V K A T N L 11 4507 139 A D P Y V V V S A 11 4508 163 L N P I F G E I L 11 4509 169 E I L E L S I S L 11 4510 184 T V A V F E H D L 11 4511 198 L I G E T H I D L 11 4512 HLA-B*08 nonamers Pos 1 2 3 4 5 6 7 8 9 score Seq. ID. NO  83 V G K F K G S F L 31 4513  32 S P K K A V A T L 29 4514  39 T L K I Y N R S L 27 4515 110 I P Q N R P I K L 25 4516 159 I P K Q L N P I F 24 4517 122 V Y V V K A T N L 22 4518 153 D T K E R Y I P K 20 4519  81 H L V G K F K G S 18 4520 155 K E R Y I P K Q L 18 4521 169 E I L E L S I S L 18 4522  24 E A E V K G T V S 17 4523  37 V A T L K I Y N R 17 4524  46 S L E E E F N H F 16 4525  61 F P L Y R G Q G G 16 4526 115 P I K L L V R V Y 16 4527 117 K L L V R V Y V V 16 4528 134 D P N G K A D P Y 16 4529 147 A G R E R Q D T K 16 4530 151 R Q D T K E R Y I 16 4531 165 P I F G E I L E L 16 4532 198 L I G E T H I D L 16 4533 HLA-B*1510 nonamers Pos 1 2 3 4 5 6 7 8 9 score Seq. ID. NO 202 T H I D L E N R F 20 4534 212 S H H R A N C G L 20 4535  92 I Y P E S E A V L 15 4536 175 I S L P A E T E L 15 4537  74 G E E E G S G H L 14 4538  80 G H L V G K F K G 14 4539  32 S P K K A V A T L 13 4540  39 T L K I Y N R S L 13 4541  55 E D W L N V F P L 13 4542 110 I P Q N R P I K L 13 4543 165 P I F G E I L E L 13 4544 184 T V A V F E H D L 13 4545  4 P G D S D G V N L 12 4546 111 P Q N R P I K L L 12 4547 169 E I L E L S I S L 12 4548 190 H D L V G S D D L 12 4549  50 E F N H F E D W L 11 4550  52 N H F E D W L N V 11 4551 122 V Y V V K A T N L 11 4552 155 K E R Y I P K Q L 11 4553 180 E T E L T V A V F 11 4554 189 E H D L V G S D D 11 4555 198 L I G E T H I D L 11 4556 213 H H R A N C G L A 11 4557  53 H F E D W L N V F 10 4558  83 V G K F K G S F L 10 4559  93 Y P E S E A V L F 10 4560 159 I P K Q L N P I F 10 4561 163 L N P I F G E I L 10 4562 HLA-B*2705 nonamers Pos 1 2 3 4 5 6 7 8 9 score Seq. ID. NO 113 N R P I K L L V R 24 4563 150 E R Q D T K E R Y 21 4564 165 P I F G E I L E L 21 4565  37 V A T L K I Y N R 18 4566  45 R S L E E E F N H 18 4567 148 G R E R Q D T K E 18 4568  74 G E E E G S G H L 17 4569  78 G S G H L V G K F 17 4570  84 G K F K G S F L I 17 4571 122 V Y V V K A T N L 17 4572 149 R E R Q D T K E R 17 4573 169 E I L E L S I S L 17 4574 175 I S L P A E T E L 17 4575 100 L F S E P Q I S R 16 4576 106 I S R G I P Q N R 16 4577 107 S R G I P Q N R P 16 4578 109 G I P Q N R P I K 16 4579 159 I P K Q L N P I F 16 4580 202 T H I D L E N R F 16 4581 208 N R F Y S H H R A 16 4582  20 Q D Q G E A E V K 15 4583  27 V K G T V S P K K 15 4584  32 S P K K A V A T L 15 4585  92 I Y P E S E A V L 15 4586 147 A G R E R Q D T K 15 4587 190 H D L V G S D D L 15 4588 216 A N C G L A S Q Y 15 4589 228 V Q Q G P Q E P F 15 4590  26 E V K G T V S P K 14 4591  33 P K K A V A T L K 14 4592  64 Y R G Q G G Q D G 14 4593  73 G G E E E G S G H 14 4594  77 E G S G H L V G K 14 4595  82 L V G K F K G S F 14 4596  85 K F K G S F L I Y 14 4597 108 R G I P Q N R P I 14 4598 110 I P Q N R P I K L 14 4599 111 P Q N R P I K L L 14 4600 118 L L V R V Y V V K 14 4601 141 P Y V V V S A G R 14 4602 155 K E R Y I P K Q L 14 4603 156 E R Y I P K Q L N 14 4604 180 E T E L T V A V F 14 4605  4 P G D S D G V N L 13 4606  5 G D S D G V N L I 13 4607  46 S L E E E F N H F 13 4608  53 H F E D W L N V F 13 4609  93 Y P E S E A V L F 13 4610 114 R P I K L L V R V 13 4611 120 V R V Y V V K A T 13 4612 157 R Y I P K Q L N P 13 4613 201 E T H I D L E N R 13 4614  35 K A V A T L K I Y 12 4615  39 T L K I Y N R S L 12 4616  43 Y N R S L E E E F 12 4617  44 N R S L E E E F N 12 4618  55 E D W L N V F P L 12 4619  56 D W L N V F P L Y 12 4620  79 S G H L V G K F K 12 4621  83 V G K F K G S F L 12 4622  98 A V L F S E P Q I 12 4623 115 P I K L L V R V Y 12 4624 134 D P N G K A D P Y 12 4625 143 V V V S A G R E R 12 4626 153 D T K E R Y I P K 12 4627 196 D D L I G E T H I 12 4628 198 L I G E T H I D L 12 4629 HLA-B*2709 nonamers Pos 1 2 3 4 5 6 7 8 9 score Seq. ID. NO 114 R P I K L L V R V 15 4630  4 P G D S D G V N L 14 4631 108 R G I P Q N R P I 14 4632 117 K L L V R V Y V V 14 4633 155 K E R Y I P K Q L 14 4634 175 I S L P A E T E L 14 4635  29 G T V S P K K A V 13 4636  52 N H F E D W L N V 13 4637  74 G E E E G S G H L 13 4638  84 G K F K G S F L I 13 4639  98 A V L F S E P Q I 13 4640 122 V Y V V K A T N L 13 4641 148 G R E R Q D T K E 13 4642 165 P I F G E I L E L 13 4643 208 N R F Y S H H R A 13 4644  5 G D S D G V N L I 12 4645  78 G S G H L V G K F 12 4646 116 I K L L V R V Y V 12 4647 120 V R V Y V V K A T 12 4648 137 G K A D P Y V V V 12 4649 151 R Q D T K E R Y I 12 4650 156 E R Y I P K Q L N 12 4651 169 E I L E L S I S L 12 4652 190 H D L V G S D D L 12 4653  11 N L I S M V G E I 11 4654  23 G E A E V K G T V 11 4655  32 S P K K A V A T L 11 4656  34 K K A V A T L K I 11 4657  55 E D W L N V F P L 11 4658  91 L I Y P E S E A V 11 4659  92 I Y P E S E A V L 11 4660  93 Y P E S E A V L F 11 4661 107 S R G I P Q N R P 11 4662 110 I P Q N R P I K L 11 4663 112 Q N R P I K L L V 11 4664 113 N R P I K L L V R 11 4665 150 E R Q D T K E R Y 11 4666 179 A E T E L T V A V 11 4667 214 H R A N C G L A S 11 4668 218 C G L A S Q Y E V 11 4669  39 T L K I Y N R S L 10 4670  44 N R S L E E E F N 10 4671  50 E F N H F E D W L 10 4672  64 Y R G Q G G Q D G 10 4673  83 V G K F K G S F L 10 4674 111 P Q N R P I K L L 10 4675 136 N G K A D P Y V V 10 4676 159 I P K Q L N P I F 10 4677 162 Q L N P I F G E I 10 4678 163 L N P I F G E I L 10 4679 184 T V A V F E H D L 10 4680 196 D D L I G E T H I 10 4681 198 L I G E T H I D L 10 4682 202 T H I D L E N R F 10 4683 212 S H H R A N C G L 10 4684   2 D D P G D S D G V 9 4685   8 D G V N L I S M V 9 4686  19 I Q D Q G E A E V 9 4687  43 Y N R S L E E E F 9 4688 102 S E P Q I S R G I 9 4689 135 P N G K A D P Y V 9 4690 167 F G E I L E L S I 9 4691 177 L P A E T E L T V 9 4692 180 E T E L T V A V F 9 4693 185 V A V F E H D L V 9 4694 191 D L V G S D D L I 9 4695 220 L A S Q Y E V W V 9 4696   7 S D G V N L I S M 8 4697  46 S L E E E F N H F 8 4698  53 H F E D W L N V F 8 4699  75 E E E G S G H L V 8 4700  82 L V G K F K G S F 8 4701 157 R Y I P K Q L N P 8 4702 158 Y I P K Q L N P I 8 4703 228 V Q Q G P Q E P F 8 4704  45 R S L E E E F N H 7 4705  88 G S F L I Y P E S 7 4706 209 R F Y S H H R A N 7 4707 HLA-B*5101 nonamers Pos 1 2 3 4 5 6 7 8 9 score Seq. ID. NO 177 L P A E T E L T V 26 4708 110 I P Q N R P I K L 22 4709 114 R P I K L L V R V 22 4710 140 D P Y V V V S A G 22 4711 220 L A S Q Y E V W V 22 4712  32 S P K K A V A T L 21 4713 136 N G K A D P Y V V 20 4714   3 D P G D S D G V N 19 4715   8 D G V N L I S M V 19 4716 185 V A V F E H D L V 19 4717 108 R G I P Q N R P I 18 4718 167 F G E I L E L S I 18 4719 196 D D L I G E T H I 18 4720 218 C G L A S Q Y E V 18 4721 134 D P N G K A D P Y 17 4722 138 K A D P Y V V V S 17 4723 130 L A P A D P N G K 16 4724 158 Y I P K Q L N P I 16 4725 178 P A E T E L T V A 16 4726 191 D L V G S D D L I 16 4727  24 E A E V K G T V S 15 4728  92 I Y P E S E A V L 15 4729 116 I K L L V R V Y V 15 4730 117 K L L V R V Y V V 15 4731   2 D D P G D S D G V 14 4732   5 G D S D G V N L I 14 4733  11 N L I S M V G E I 14 4734  23 G E A E V K G T V 14 4735  35 K A V A T L K I Y 14 4736  83 V G K F K G S F L 14 4737  91 L I Y P E S E A V 14 4738  93 Y P E S E A V L F 14 4739 131 A P A D P N G K A 14 4740   4 P G D S D G V N L 13 4741  34 K K A V A T L K I 13 4742  37 V A T L K I Y N R 13 4743  52 N H F E D W L N V 13 4744  61 F P L Y R G Q G G 13 4745  98 A V L F S E P Q I 13 4746 137 G K A D P Y V V V 13 4747 151 R Q D T K E R Y I 13 4748 159 I P K Q L N P I F 13 4749 part 3: MHC Class I nonamer analysis of 158P3D2 v.3 (aa 95-111, PTEREVSVRRRSGPFAL). HLA-A1 nonamers Pos 1 2 3 4 5 6 7 8 9 score Seq. ID. NO  95 P T E R E V S V R 18 4750 HLA-A26 nonamers Pos 1 2 3 4 5 6 7 8 9 score Seq. ID. NO 101 S V R R R S G P F 20 4751  99 E V S V R R R S G 17 4752  94 P T E R E V S V R 15 4753 HLA-A3 nonamers Pos 1 2 3 4 5 6 7 8 9 score Seq. ID. NO 101 S V R R R S G P F 24 4754  99 E V S V R R R S G 15 4755 HLA-B*0702 nonamers Pos 1 2 3 4 5 6 7 8 9 score Seq. ID. NO 103 R R R S G P F A L 14 4756 102 V R R R S G P F A 11 4757 HLA-B*08 nonamers Pos 1 2 3 4 5 6 7 8 9 score Seq. ID. NO 101 S V R R R S G P F 22 4758 103 R R R S G P F A L 17 4759 HLA-B*1510 nonamers Pos 1 2 3 4 5 6 7 8 9 score Seq. ID. NO 103 R R R S G P F A L 13 4760  96 E R E V S V R R R 8 4761 HLA-B*2705 nonamers Pos 1 2 3 4 5 6 7 8 9 score Seq. ID. NO 103 R R R S G P F A L 26 4762  96 E R E V S V R R R 24 4763  95 T E R E V S V R R 16 4764  94 P T E R E V S V R 14 4765 HLA-B*2709 nonamers Pos 1 2 3 4 5 6 7 8 9 score Seq. ID. NO 103 R R R S G P F A L 25 4766 part 4: MHC Class I nonamer analysis of 158P3D2 v.4 (aa 94-110, LPTEREVSIWRRSGPFA). HLA-A*0201 nonamers Pos 1 2 3 4 5 6 7 8 9 score Seq. ID. NO  94 L P T E R E V S I 15 4767 HLA-A1 nonamers Pos 1 2 3 4 5 6 7 8 9 score Seq. ID. NO  95 P T E R E V S I W 17 4768 HLA-A26 nonamers Pos 1 2 3 4 5 6 7 8 9 score Seq. ID. NO 101 S I W R R S G P F 20 4769  99 E V S I W R R S G 17 4770  95 P T E R E V S I W 15 4771 HLA-A3 nonamers Pos 1 2 3 4 5 6 7 8 9 score Seq. ID. NO 101 S I W R R S G P F 19 4772 HLA-B*0702 nonamers Pos 1 2 3 4 5 6 7 8 9 score Seq. ID. NO  94 L P T E R E V S I 18 4773 102 I W R R S G P F A 12 4774 HLA-B*08 nonamers Pos 1 2 3 4 5 6 7 8 9 score Seq. ID. NO  94 L P T E R E V S I 23 4775 101 S I W R R S G P F 20 4776 HLA-B*2705 nonamers Pos 1 2 3 4 5 6 7 8 9 score Seq. ID. NO   4 E R E V S I W R R 27 4777   3 T E R E V S I W R 14 4778 HLA-B*5101 nonamers Pos 1 2 3 4 5 6 7 8 9 score Seq. ID. NO  94 L P T E R E V S I 25 4779 part 5: MHC Class I nonamer analysis of 158P3D2 v.4 (aa 122-178, LVLQVWDYT ASLPMTSLDP WSCSYQTWCV GPGAPSSALC SWPAMGPGRG AICFAAAA) HLA-A*0201 nonamers Pos 1 2 3 4 5 6 7 8 9 score Seq. ID. NO 125 Q V W D Y T A S L 23 4780 164 A M G P G R G A I 23 4781 123 V L Q V W D Y T A 18 4782 130 T A S L P M T S L 17 4783 137 S L D P W S C S Y 16 4784 170 G A I C F A A A A 15 4785 157 S A L C S W P A M 14 4786 158 A L C S W P A M G 14 4787 132 S L P M T S L D P 13 4788 142 S C S Y Q T W C V 13 4789 151 G P G A P S S A L 13 4790 121 L V L Q V W D Y T 12 4791 129 Y T A S L P M T S 11 4792 146 Q T W C V G P G A 11 4793 149 C V G P G A P S S 11 4794 HLA-A1 nonamers Pos 1 2 3 4 5 6 7 8 9 score Seq. ID. NO  16 S L D P W S C S Y 32 4795 HLA-A26 nonamers Pos 1 2 3 4 5 6 7 8 9 score Seq. ID. NO  16 S L D P W S C S Y 22 4796   4 Q V W D Y T A S L 21 4797   8 Y T A S L P M T S 14 4798  28 C V G P G A P S S 14 4799   7 D Y T A S L P M T 13 4800   9 T A S L P M T S L 13 4801  25 Q T W C V G P G A 12 4802   1 L V L Q V W D Y T 11 4803  14 M T S L D P W S C 11 4804  36 S A L C S W P A M 11 4805  37 A L C S W P A M G 11 4806 HLA-A3 nonamers Pos 1 2 3 4 5 6 7 8 9 score Seq. ID. NO  16 S L D P W S C S Y 22 4807  28 C V G P G A P S S 20 4808   4 Q V W D Y T A S L 18 4809  37 A L C S W P A M G 18 4810   2 V L Q V W D Y T A 14 4811  11 S L P M T S L D P 14 4812   1 L V L Q V W D Y T 13 4813 166 G P G R G A I C F 12 4814 131 A S L P M T S L D 11 4815 164 A M G P G R G A I 11 4816 HLA-B*0702 nonamers Pos 1 2 3 4 5 6 7 8 9 score Seq. ID. NO 151 G P G A P S S A L 26 4817 166 G P G R G A I C F 17 4818 130 T A S L P M T S L 16 4819 139 D P W S C S Y Q T 16 4820 154 A P S S A L C S W 14 4821 163 P A M G P G R G A 13 4822 HLA-B*08 nonamers Pos 1 2 3 4 5 6 7 8 9 score Seq. ID. NO 151 G P G A P S S A L 18 4823 130 T A S L P M T S L 15 4824 166 G P G R G A I C F 13 4825 125 Q V W D Y T A S L 10 4826 HLA-B*1510 nonamers Pos 1 2 3 4 5 6 7 8 9 score Seq. ID. NO 130 T A S L P M T S L 14 4827 151 G P G A P S S A L 13 4828 125 Q V W D Y T A S L 11 4829 157 S A L C S W P A M 8 4830 166 G P G R G A I C F 8 4831 HLA-B*2705 nonamers Pos 1 2 3 4 5 6 7 8 9 score Seq. ID. NO 166 G P G R G A I C F 17 4832 151 G P G A P S S A L 16 4833 130 T A S L P M T S L 15 4834 168 G R G A I C F A A 14 4835 125 Q V W D Y T A S L 12 4836 127 W D Y T A S L P M 12 4837 157 S A L C S W P A M 12 4838 137 S L D P W S C S Y 11 4839 161 S W P A M G P G R 11 4840 164 A M G P G R G A I 10 4841 HLA-B*2709 nonamers Pos 1 2 3 4 5 6 7 8 9 score Seq. ID. NO 168 G R G A I C F A A 14 4842 151 G P G A P S S A L 13 4843 146 G P G R G A I C F 12 4844 127 W D Y T A S L P M 11 4845 157 S A L C S W P A M 11 4846 125 Q V W D Y T A S L 10 4847 130 T A S L P M T S L 10 4848 161 A M G P G R G A I 10 4849 142 S C S Y Q T W C V 8 4850 HLA-B*4402 nonamers Pos 1 2 3 4 5 6 7 8 9 score Seq. ID. NO 164 A M G P G R G A I 17 4851 137 S L D P W S C S Y 15 4852 154 A P S S A L C S W 15 4853 133 L P M T S L D P W 13 4854 166 G P G R G A I C F 13 4855 125 Q V W D Y T A S L 12 4856 130 T A S L P M T S L 12 4857 140 P W S C S Y Q T W 12 4858 151 G P G A P S S A L 12 4859 131 A S L P M T S L D 9 4860 HLA-B*5101 nonamers Pos 1 2 3 4 5 6 7 8 9 score Seq. ID. NO 130 T A S L P M T S L 18 4861 151 G P G A P S S A L 17 4862 133 L P M T S L D P W 15 4863 139 D P W S C S Y Q T 15 4864 153 G A P S S A L C S 14 4865 157 S A L C S W P A M 13 4866 162 W P A M G P G R G 12 4867 163 P A M G P G R G A 12 4868 166 G P G R G A I C F 12 4869 154 A P S S A L C S W 11 4870 170 G A I C F A A A A 11 4871 150 V G P G A P S S A 10 4872 164 A M G P G R G A I 10 4873 125 Q V W D Y T A S L 9 4874 165 M G P G R G A I C 9 4875

TABLE XIXB MHC Class I Analysis of 158P3D2 (decamers) part 1: MHC Class I decamer analysis of 158P3D2 v.1 (aa 1-328) Listed are scores which correlate with the ligation strength to a defined HLA type for a sequence of amino acids. The algorithms used are based on the book “MHC Ligands and Peptide Motifs” by H. G. Rammensee, J. Bachmann and S. Stevanovic. The probability of being processed and presented is given in order to predict T-cell epitopes. HLA-A*0201 decamers Pos 1 2 3 4 5 6 7 8 9 0 score Seq. ID. NO 296 V L L L L V L L T V 30 4876 301 V L L T V F L L L V 28 4877  93 Y L P T E R E V S V 26 4878 294 T L V L L L L V L L 26 4879 298 L L L V L L T V F L 26 4880 299 L L V L L T V F L L 26 4881 312 Y T I P G Q I S Q V 24 4882 151 G A R G P E L C S V 23 4883  31 V I W N T E D V V L 22 4884 236 V E A E F E L L T V 22 4885 286 F I W R R Y W R T L 22 4886 140 S L E L Q L P D M V 21 4887 293 R T L V L L L L V L 21 4888 132 I S A N D F L G S L 20 4889 179 L R G W W P V V K L 20 4890 123 V L Q V W D Y D R I 19 4891 148 M V R G A R G P E L 19 4892 300 L V L L T V F L L L 19 4893 223 T D M G G N V Y I L 18 4894 297 L L L L V L L T V F 18 4895 313 T I P G Q I S Q V I 18 4896 317 Q I S Q V I F R P L 18 4897  30 V V I W N T E D V V 17 4898 230 Y I L T G K V E A E 17 4899 231 I L T G K V E A E F 17 4900 289 R R Y W R T L V L L 17 4901 291 Y W R T L V L L L L 17 4902 295 L V L L L L V L L T 17 4903 308 L L V F Y T I P G Q 17 4904   4 D I F P Q D V P A P 16 4905  22 Q P I S Y E L R V V 16 4906  45 P L T G E M S S D I 16 4907  54 I Y V K S W V K G L 16 4908 187 K L K E A E D V E R 16 4909 271 T S F N W F V N P L 16 4910 290 R Y W R T L V L L L 16 4911 302 L L T V F L L L V F 16 4912  39 V L D D E N P L T G 15 4913 114 A E F R Q P A V L V 15 4914 117 R Q P A V L V L Q V 15 4915 137 F L G S L E L Q L P 15 4916 143 L Q L P D M V R G A 15 4917 222 F T D M G G N V Y I 15 4918 244 T V E E A E K R P V 15 4919 HLA-A*0203 decamers Pos 1 2 3 4 5 6 7 8 9 0 score Seq. ID. NO 194 V E R E A Q E A Q A 18 4920   3 I D I F P Q D V P A 10 4921 101 S V W R R S G P F A 10 4922 105 R S G P F A L E E A 10 4923 111 L E E A E F R Q P A 10 4924 125 Q V W D Y D R I S A 10 4925 143 L Q L P D M V R G A 10 4926 154 G P E L C S V Q L A 10 4927 158 C S V Q L A R N G A 10 4928 182 W W P V V K L K E A 10 4929 189 K E A E D V E R E A 10 4930 192 E D V E R E A Q E A 10 4931 229 V Y I L T G K V E A 10 4932 239 E F E L L T V E E A 10 4933   4 D I F P Q D V P A P  9 4934 102 V W R R S G P F A L  9 4935 106 S G P F A L E E A E  9 4936 112 E E A E F R Q P A V  9 4937 126 V W D Y D R I S A N  9 4938 144 Q L P D M V R G A R  9 4939 155 P E L C S V Q L A R  9 4940 159 S V Q L A R N G A G  9 4941 183 W P V V K L K E A E  9 4942 190 E A E D V E R E A Q  9 4943 193 D V E R E A Q E A Q  9 4944 195 E R E A Q E A Q A G  9 4945 230 Y I L T G K V E A E  9 4946 240 F E L L T V E E A E  9 4947 HLA-A1 decamers Pos 1 2 3 4 5 6 7 8 9 0 score Seq. ID. NO  17 D I K P R Q P I S Y 23 4948  46 L T G E M S S D I Y 21 4949 303 L T V F L L L V F Y 21 4950  69 E T D V H F N S L T 19 4951  39 V L D D E N P L T G 18 4952 222 F T D M G G N V Y I 18 4953 235 K V E A E F E L L T 18 4954  25 S Y E L R V V I W N 17 4955 120 A V L V L Q V W D Y 17 4956 134 A N D F L G S L E L 17 4957 221 E F T D M G G N V Y 17 4958  34 N T E D V V L D D E 16 4959  51 S S D I Y V K S W V 16 4960  84 N W R F V F R F D Y 16 4961  95 P T E R E V S V W R 16 4962 110 A L E E A E F R Q P 15 4963 282 T F V F F I W R R Y 15 4964  47 T G E M S S D I Y V 14 4965 140 S L E L Q L P D M V 14 4966 198 A Q E A Q A G K K K 14 4967 259 Q P E P L E K P S R 14 4968 262 P L E K P S R P K T 14 4969 154 G P E L C S V Q L A 13 4970 216 R P E D L E F T D M 13 4971 245 V E E A E K R P V G 13 4972 247 E A E K R P V G K G 13 4973 293 R T L V L L L L V L 13 4974  78 T G E G N F N W R F 12 4975 181 G W W P V V K L K E 12 4976 312 Y T I P G Q I S Q V 12 4977   2 W I D I F P Q D V P 11 4978  15 P V D I K P R Q P I 11 4979  62 G L E H D K Q E T D 11 4980  64 E H D K Q E T D V H 11 4981 111 L E E A E F R Q P A 11 4982 113 E A E F R Q P A V L 11 4983 126 V W D Y D R I S A N 11 4984 145 L P D M V R G A R G 11 4985 166 G A G P R C N L F R 11 4986 188 L K E A E D V E R E 11 4987 190 E A E D V E R E A Q 11 4988 191 A E D V E R E A Q E 11 4989 217 P E D L E F T D M G 11 4990 219 D L E F T D M G G N 11 4991 237 E A E F E L L T V E 11 4992 239 E F E L L T V E E A 11 4993 300 L V L L T V F L L L 11 4994 HLA-A26 decamers Pos 1 2 3 4 5 6 7 8 9 0 score Seq. ID. NO  17 D I K P R Q P I S Y 30 4995   4 D I F P Q D V P A P 26 4996 115 E F R Q P A V L V L 25 4997 303 L T V F L L L V F Y 25 4998  37 D V V L D D E N P L 24 4999 120 A V L V L Q V W D Y 24 5000 136 D F L G S L E L Q L 23 5001 221 E F T D M G G N V Y 23 5002 317 Q I S Q V I F R P L 23 5003  46 L T G E M S S D I Y 22 5004 148 M V R G A R G P E L 22 5005 231 I L T G K V E A E F 22 5006 276 F V N P L K T F V F 22 5007 293 R T L V L L L L V L 22 5008 297 L L L L V L L T V F 22 5009 300 L V L L T V F L L L 22 5010 302 L L T V F L L L V F 22 5011  71 D V H F N S L T G E 21 5012  82 N F N W R F V F R F 21 5013 156 E L C S V Q L A R N 21 5014 294 T L V L L L L V L L 21 5015 142 E L Q L P D M V R G 20 5016 299 L L V L L T V F L L 20 5017 312 Y T I P G Q I S Q V 20 5018  31 V I W N T E D V V L 19 5019 219 D L E F T D M G G N 19 5020 264 E K P S R P K T S F 19 5021   9 D V P A P P P V D I 18 5022  53 D I Y V K S W V K G 18 5023  69 E T D V H F N S L T 18 5024  99 E V S V W R R S G P 18 5025 286 F I W R R Y W R T L 18 5026 128 D Y D R I S A N D F 17 5027 193 D V E R E A Q E A Q 17 5028 239 E F E L L T V E E A 17 5029 281 K T F V F F I W R R 17 5030 282 T F V F F I W R R Y 17 5031 298 L L L V L L T V F L 17 5032  77 L T G E G N F N W R 16 5033  80 E G N F N W R F V F 16 5034  87 F V F R F D Y L P T 16 5035 241 E L L T V E E A E K 16 5036 267 S R P K T S F N W F 16 5037 270 K T S F N W F V N P 16 5038 274 N W F V N P L K T F 16 5039 277 V N P L K T F V F F 16 5040 304 T V F L L L V F Y T 16 5041  34 N T E D V V L D D E 15 5042  41 D D E N P L T G E M 15 5043 110 A L E E A E F R Q P 15 5044 113 E A E F R Q P A V L 15 5045 131 R I S A N D F L G S 15 5046 139 G S L E L Q L P D M 15 5047 230 Y I L T G K V E A E 15 5048 HLA-A3 decamers Pos 1 2 3 4 5 6 7 8 9 0 score Seq. ID. NO 178 R L R G W W P V V K 38 5049 241 E L L T V E E A E K 25 5050 161 Q L A R N G A G P R 24 5051 187 K L K E A E D V E R 23 5052 228 N V Y I L T G K V E 22 5053 297 L L L L V L L T V F 22 5054  17 D I K P R Q P I S Y 21 5055 120 A V L V L Q V W D Y 21 5056 231 I L T G K V E A E F 21 5057 276 F V N P L K T F V F 21 5058 302 L L T V F L L L V F 21 5059 144 Q L P D M V R G A R 20 5060 296 V L L L L V L L T V 20 5061  39 V L D D E N P L T G 19 5062 HLA-B*0702 decamers Pos 1 2 3 4 5 6 7 8 9 0 score Seq. ID. NO  19 K P R Q P I S Y E L 23 5063 314 I P G Q I S Q V I F 20 5064 216 R P E D L E F T D M 19 5065 278 N P L K T F V F F I 19 5066 107 G P F A L E E A E F 18 5067 268 R P K T S F N W F V 18 5068  22 Q P I S Y E L R V V 17 5069 115 E F R Q P A V L V L 17 5070 154 G P E L C S V Q L A 17 5071 288 W R R Y W R T L V L 16 5072 134 A N D F L G S L E L 15 5073 148 M V R G A R G P E L 15 5074 265 K P S R P K T S F N 15 5075  12 A P P P V D I K P R 14 5076 136 D F L G S L E L Q L 14 5077 164 R N G A G P R C N L 14 5078 179 L R G W W P V V K L 14 5079 211 R R R K G R P E D L 14 5080 223 T D M G G N V Y I L 14 5081 251 R P V G K G R K Q P 14 5082 290 R Y W R T L V L L L 14 5083 291 Y W R T L V L L L L 14 5084 293 R T L V L L L L V L 14 5085 298 L L L V L L T V F L 14 5086 317 Q I S Q V I F R P L 14 5087  10 V P A P P P V D I K 13 5088  31 V I W N T E D V V L 13 5089  54 I Y V K S W V K G L 13 5090 102 V W R R S G P F A L 13 5091 113 E A E F R Q P A V L 13 5092 129 Y D R I S A N D F L 13 5093 153 R G P E L C S V Q L 13 5094 168 G P R C N L F R C R 13 5095 289 R R Y W R T L V L L 13 5096 300 L V L L T V F L L L 13 5097   6 F P Q D V P A P P P 12 5098  23 P I S Y E L R V V I 12 5099  94 L P T E R E V S V W 12 5100 118 Q P A V L V L Q V W 12 5101 132 I S A N D F L G S L 12 5102 145 L P D M V R G A R G 12 5103 254 G K G R K Q P E P L 12 5104 259 Q P E P L E K P S R 12 5105 261 E P L E K P S R P K 12 5106 271 T S F N W F V N P L 12 5107 294 T L V L L L L V L L 12 5108 HLA-B*4402 decamers Pos 1 2 3 4 5 6 7 8 9 0 score Seq. ID. NO  68 Q E T D V H F N S L 23 5109 114 A E F R Q P A V L V 19 5110 238 A E F E L L T V E E 17 5111 263 L E K P S R P K T S 17 5112 274 N W F V N P L K T F 17 5113 248 A E K R P V G K G R 16 5114  17 D I K P R Q P I S Y 15 5115  26 Y E L R V V I W N T 15 5116  48 G E M S S D I Y V K 15 5117  50 M S S D I Y V K S W 15 5118 115 E F R Q P A V L V L 15 5119 120 A V L V L Q V W D Y 15 5120 134 A N D F L G S L E L 15 5121 191 A E D V E R E A Q E 15 5122 221 E F T D M G G N V Y 15 5123 271 T S F N W F V N P L 15 5124 276 F V N P L K T F V F 15 5125 300 L V L L T V F L L L 15 5126  80 E G N F N W R F V F 14 5127 102 V W R R S G P F A L 14 5128 112 E E A E F R Q P A V 14 5129 113 E A E F R Q P A V L 14 5130 128 D Y D R I S A N D F 14 5131 136 D F L G S L E L Q L 14 5132 155 P E L C S V Q L A R 14 5133 165 N G A G P R C N L F 14 5134 240 F E L L T V E E A E 14 5135 246 E E A E K R P V G K 14 5136 267 S R P K T S F N W F 14 5137 277 V N P L K T F V F F 14 5138 283 F V F F I W R R Y W 14 5139 290 R Y W R T L V L L L 14 5140 291 Y W R T L V L L L L 14 5141 293 R T L V L L L L V L 14 5142 294 T L V L L L L V L L 14 5143 297 L L L L V L L T V F 14 5144  31 V I W N T E D V V L 13 5145  42 D E N P L T G E M S 13 5146  54 I Y V K S W V K G L 13 5147  85 W R F V F R F D Y L 13 5148 153 R G P E L C S V Q L 13 5149 179 L R G W W P V V K L 13 5150 199 Q E A Q A G K K K R 13 5151 217 P E D L E F T D M G 13 5152 220 L E F T D M G G N V 13 5153 223 T D M G G N V Y I L 13 5154 236 V E A E F E L L T V 13 5155 260 P E P L E K P S R P 13 5156 264 E K P S R P K T S F 13 5157 266 P S R P K T S F N W 13 5158 286 F I W R R Y W R T L 13 5159 288 W R R Y W R T L V L 13 5160 289 R R Y W R T L V L L 13 5161 298 L L L V L L T V F L 13 5162 299 L L V L L T V F L L 13 5163 302 L L T V F L L L V F 13 5164 317 Q I S Q V I F R P L 13 5165  12 A P P P V D I K P R 12 5166  23 P I S Y E L R V V I 12 5167  24 I S Y E L R V V I W 12 5168  37 D V V L D D E N P L 12 5169  74 F N S L T G E G N F 12 5170  76 S L T G E G N F N W 12 5171  79 G E G N F N W R F V 12 5172  82 N F N W R F V F R F 12 5173  84 N W R F V F R F D Y 12 5174  94 L P T E R E V S V W 12 5175  98 R E V S V W R R S G 12 5176 107 G P F A L E E A E F 12 5177 118 Q P A V L V L Q V W 12 5178 132 I S A N D F L G S L 12 5179 141 L E L Q L P D M V R 12 5180 170 R C N L F R C R R L 12 5181 173 L F R C R R L R G W 12 5182 174 F R C R R L R G W W 12 5183 189 K E A E D V E R E A 12 5184 213 R K G R P E D L E F 12 5185 234 G K V E A E F E L L 12 5186 245 V E E A E K R P V G 12 5187 254 G K G R K Q P E P L 12 5188 279 P L K T F V F F I W 12 5189 303 L T V F L L L V F Y 12 5190 305 V F L L L V F Y T I 12 5191 309 L V F Y T I P G Q I 12 5192   9 D V P A P P P V D I 11 5193  19 K P R Q P I S Y E L 11 5194  35 T E D V V L D D E N 11 5195  63 L E H D K Q E T D V 11 5196  65 H D K Q E T D V H F 11 5197  78 T G E G N F N W R F 11 5198  96 T E R E V S V W R R 11 5199 111 L E E A E F R Q P A 11 5200 148 M V R G A R G P E L 11 5201 164 R N G A G P R C N L 11 5202 194 V E R E A Q E A Q A 11 5203 211 R R R K G R P E D L 11 5204 231 I L T G K V E A E F 11 5205 278 N P L K T F V F F I 11 5206 282 T F V F F I W R R Y 11 5207 313 T I P G Q I S Q V I 11 5208 314 I P G Q I S Q V I F 11 5209 part 2: MHC Class I decamer analysis of 158P3D2 v.2a (aa 1-236). HLA-A*0201 decamers Pos 1 2 3 4 5 6 7 8 9 0 score Seq. ID. NO  91 L I Y P E S E A V L 25 5210 176 S L P A E T E L T V 25 5211 219 G L A S Q Y E V W V 25 5212 118 L L V R V Y V V K A 24 5213  90 F L I Y P E S E A V 23 5214 162 Q L N P I F G E I L 23 5215 197 D L I G E T H I D L 23 5216 174 S I S L P A E T E L 22 5217 109 G I P Q N R P I K L 21 5218  18 E I Q D Q G E A E V 20 5219  38 A T L K I Y N R S L 20 5220  31 V S P K K A V A T L 19 5221 116 I K L L V R V Y V V 19 5222 138 K A D P Y V V V S A 19 5223 164 N P I F G E I L E L 18 5224  10 V N L I S M V G E I 17 5225 157 R Y I P K Q L N P I 17 5226 183 L T V A V F E H D L 17 5227   7 S D G V N L I S M V 16 5228  41 K I Y N R S L E E E 16 5229  57 W L N V F P L Y R G 16 5230  82 L V G K F K G S F L 16 5231 113 N R P I K L L V R V 16 5232 115 P I K L L V R V Y V 16 5233 129 N L A P A D P N G K 16 5234 170 I L E L S I S L P A 16 5235 184 T V A V F E H D L V 16 5236 186 A V F E H D L V G S 16 5237 198 L I G E T H I D L E 16 5238  54 F E D W L N V F P L 15 5239 110 I P Q N R P I K L L 15 5240 117 K L L V R V Y V V K 15 5241 121 R V Y V V K A T N L 15 5242 166 I F G E I L E L S I 15 5243 168 G E I L E L S I S L 15 5244 172 E L S I S L P A E T 15 5245  46 S L E E E F N H F E 14 5246  81 H L V G K F K G S F 14 5247  99 V L F S E P Q I S R 14 5248 119 L V R V Y V V K A T 14 5249 124 V V K A T N L A P A 14 5250 177 L P A E T E L T V A 14 5251   1 M D D P G D S D G V 13 5252  30 T V S P K K A V A T 13 5253  36 A V A T L K I Y N R 13 5254  74 G E E E G S G H L V 13 5255 134 D P N G K A D P Y V 13 5256 161 K Q L N P I F G E I 13 5257 169 E I L E L S I S L P 13 5258 178 P A E T E L T V A V 13 5259 211 Y S H H R A N C G L 13 5260 217 N C G L A S Q Y E V 13 5261   3 D P G D S D G V N L 12 5262  11 N L I S M V G E I Q 12 5263  14 S M V G E I Q D Q G 12 5264  73 G G E E E G S G H L 12 5265 130 L A P A D P N G K A 12 5266 165 P I F G E I L E L S 12 5267 191 D L V G S D D L I G 12 5268 193 V G S D D L I G E T 12 5269 HLA-A*0203 decamers Pos 1 2 3 4 5 6 7 8 9 0 score Seq. ID. NO  29 G T V S P K K A V A 18 5270 124 V V K A T N L A P A 18 5271  16 V G E I Q D Q G E A 10 5272  27 V K G T V S P K K A 10 5273  89 S F L I Y P E S E A 10 5274 118 L L V R V Y V V K A 10 5275 122 V Y V V K A T N L A 10 5276 130 L A P A D P N G K A 10 5277 138 K A D P Y V V V S A 10 5278 170 I L E L S I S L P A 10 5279 177 L P A E T E L T V A 10 5280 207 E N R F Y S H H R A 10 5281 212 S H H R A N C G L A 10 5282  17 G E I Q D Q G E A E  9 5283  28 K G T V S P K K A V  9 5284  30 T V S P K K A V A T  9 5285  90 F L I Y P E S E A V  9 5286 119 L V R V Y V V K A T  9 5287 123 Y V V K A T N L A P  9 5288 125 V K A T N L A P A D  9 5289 131 A P A D P N G K A D  9 5290 139 A D P Y V V V S A G  9 5291 171 L E L S I S L P A E  9 5292 178 P A E T E L T V A V  9 5293 208 N R F Y S H H R A N  9 5294 213 H H R A N C G L A S  9 5295 HLA-A1 decamers Pos 1 2 3 4 5 6 7 8 9 0 score Seq. ID. NO  84 G K F K G S F L I Y 24 5296  55 E D W L N V F P L Y 20 5297   6 D S D G V N L I S M 19 5298  93 Y P E S E A V L F S 19 5299 101 F S E P Q I S R G I 19 5300  75 E E E G S G H L V G 18 5301  95 E S E A V L F S E P 17 5302 114 R P I K L L V R V Y 17 5303 170 I L E L S I S L P A 17 5304 133 A D P N G K A D P Y 16 5305 180 E T E L T V A V F E 16 5306 199 I G E T H I D L E N 16 5307 202 T H I D L E N R F Y 16 5308  34 K K A V A T L K I Y 15 5309 138 K A D P Y V V V S A 15 5310 149 R E R Q D T K E R Y 15 5311 194 G S D D L I G E T H 15 5312 215 R A N C G L A S Q Y 15 5313   1 M D D P G D S D G V 14 5314  46 S L E E E F N H F E 14 5315 132 P A D P N G K A D P 14 5316   4 P G D S D G V N L I 13 5317  48 E E E F N H F E D W 13 5318  74 G E E E G S G H L V 13 5319  54 F E D W L N V F P L 12 5320 187 V F E H D L V G S D 12 5321 195 S D D L I G E T H I 12 5322 HLA-A26 decamers Pos 1 2 3 4 5 6 7 8 9 0 score Seq. ID. NO 201 E T H I D L E N R F 27 5323 197 D L I G E T H I D L 26 5324 153 D T K E R Y I P K Q 25 5325  77 E G S G H L V G K F 23 5326 158 Y I P K Q L N P I F 23 5327 169 E I L E L S I S L P 23 5328   6 D S D G V N L I S M 22 5329  26 E V K G T V S P K K 21 5330  55 E D W L N V F P L Y 21 5331  81 H L V G K F K G S F 21 5332  91 L I Y P E S E A V L 21 5333 109 G I P Q N R P I K L 21 5334 227 W V Q Q G P Q E P F 21 5335  38 A T L K I Y N R S L 20 5336  82 L V G K F K G S F L 20 5337 186 A V F E H D L V G S 20 5338 205 D L E N R F Y S H H 20 5339  18 E I Q D Q G E A E V 19 5340 121 R V Y V V K A T N L 19 5341 174 S I S L P A E T E L 19 5342 182 E L T V A V F E H D 19 5343  52 N H F E D W L N V F 18 5344 162 Q L N P I F G E I L 18 5345 165 P I F G E I L E L S 18 5346 183 L T V A V F E H D L 18 5347 225 E V W V Q Q G P Q E 18 5348   3 D P G D S D G V N L 17 5349  45 R S L E E E F N H F 17 5350  84 G K F K G S F L I Y 17 5351 114 R P I K L L V R V Y 17 5352 179 A E T E L T V A V F 17 5353 180 E T E L T V A V F E 17 5354   9 G V N L I S M V G E 16 5355  36 A V A T L K I Y N R 16 5356  49 E E F N H F E D W L 16 5357 124 V V K A T N L A P A 16 5358 172 E L S I S L P A E T 16 5359 191 D L V G S D D L I G 16 5360 192 L V G S D D L I G E 16 5361 198 L I G E T H I D L E 16 5362  31 V S P K K A V A T L 15 5363  34 K K A V A T L K I Y 15 5364  41 K I Y N R S L E E E 15 5365  59 N V F P L Y R G Q G 15 5366 119 L V R V Y V V K A T 15 5367 164 N P I F G E I L E L 15 5368 189 E H D L V G S D D L 15 5369  15 M V G E I Q D Q G E 14 5370  30 T V S P K K A V A T 14 5371  92 I Y P E S E A V L F 14 5372 100 L F S E P Q I S R G 14 5373 118 L L V R V Y V V K A 14 5374  21 D Q G E A E V K G T 13 5375  54 F E D W L N V F P L 13 5376  57 W L N V F P L Y R G 13 5377  76 E E G S G H L V G K 13 5378  85 K F K G S F L I Y P 13 5379 117 K L L V R V Y V V K 13 5380 202 T H I D L E N R F Y 13 5381 HLA-A3 decamers Pos 1 2 3 4 5 6 7 8 9 0 score Seq. ID. NO 117 K L L V R V Y V V K 33 5382 129 N L A P A D P N G K 25 5383  62 P L Y R G Q G G Q D 24 5384  26 E V K G T V S P K K 23 5385  91 L I Y P E S E A V L 22 5386 121 R V Y V V K A T N L 22 5387  30 T V S P K K A V A T 21 5388 108 R G I P Q N R P I K 21 5389 176 S L P A E T E L T V 20 5390  19 I Q D Q G E A E V K 19 5391  81 H L V G K F K G S F 19 5392 112 Q N R P I K L L V R 19 5393 215 R A N C G L A S Q Y 19 5394  36 A V A T L K I Y N R 18 5395  59 N V F P L Y R G Q G 18 5396 105 Q I S R G I P Q N R 18 5397 146 S A G R E R Q D T K 18 5398 162 Q L N P I F G E I L 18 5399 186 A V F E H D L V G S 18 5400 205 D L E N R F Y S H H 18 5401 114 R P I K L L V R V Y 17 5402 118 L L V R V Y V V K A 17 5403 142 Y V V V S A G R E R 17 5404  11 N L I S M V G E I Q 16 5405  25 A E V K G T V S P K 16 5406  32 S P K K A V A T L K 16 5407  39 T L K I Y N R S L E 16 5408  41 K I Y N R S L E E E 16 5409  82 L V G K F K G S F L 16 5410  98 A V L F S E P Q I S 16 5411  99 V L F S E P Q I S R 16 5412 124 V V K A T N L A P A 16 5413 170 I L E L S I S L P A 16 5414 219 G L A S Q Y E V W V 16 5415 225 E V W V Q Q G P Q E 16 5416 HLA-B*0702 decamers Pos 1 2 3 4 5 6 7 8 9 0 score Seq. ID. NO   3 D P G D S D G V N L 23 5417 164 N P I F G E I L E L 22 5418 110 I P Q N R P I K L L 21 5419 134 D P N G K A D P Y V 19 5420 177 L P A E T E L T V A 19 5421  93 Y P E S E A V L F S 14 5422 114 R P I K L L V R V Y 14 5423 131 A P A D P N G K A D 14 5424  31 V S P K K A V A T L 13 5425  38 A T L K I Y N R S L 13 5426  54 F E D W L N V F P L 13 5427  82 L V G K F K G S F L 13 5428  91 L I Y P E S E A V L 13 5429 174 S I S L P A E T E L 13 5430  30 T V S P K K A V A T 12 5431  32 S P K K A V A T L K 12 5432  77 E G S G H L V G K F 12 5433 103 E P Q I S R G I P Q 12 5434 121 R V Y V V K A T N L 12 5435 138 K A D P Y V V V S A 12 5436 159 I P K Q L N P I F G 12 5437 189 E H D L V G S D D L 12 5438 197 D L I G E T H I D L 12 5439  49 E E F N H F E D W L 11 5440 140 D P Y V V V S A G R 11 5441 162 Q L N P I F G E I L 11 5442 179 A E T E L T V A V F 11 5443 183 L T V A V F E H D L 11 5444 HLA-B*4402 decamers Pos 1 2 3 4 5 6 7 8 9 0 score Seq. ID. NO  49 E E F N H F E D W L 25 5445 168 G E I L E L S I S L 25 5446 179 A E T E L T V A V F 25 5447  48 E E E F N H F E D W 23 5448  54 F E D W L N V F P L 22 5449 149 R E R Q D T K E R Y 20 5450 164 N P I F G E I L E L 18 5451 110 I P Q N R P I K L L 17 5452  52 N H F E D W L N V F 16 5453  77 E G S G H L V G K F 16 5454 114 R P I K L L V R V Y 16 5455 133 A D P N G K A D P Y 16 5456 157 R Y I P K Q L N P I 16 5457  17 G E I Q D Q G E A E 15 5458  38 A T L K I Y N R S L 15 5459  55 E D W L N V F P L Y 15 5460  75 E E E G S G H L V G 15 5461 154 T K E R Y I P K Q L 15 5462 197 D L I G E T H I D L 15 5463 202 T H I D L E N R F Y 15 5464  25 A E V K G T V S P K 14 5465  34 K K A V A T L K I Y 14 5466  76 E E G S G H L V G K 14 5467  84 G K F K G S F L I Y 14 5468  91 L I Y P E S E A V L 14 5469  92 I Y P E S E A V L F 14 5470 109 G I P Q N R P I K L 14 5471 171 L E L S I S L P A E 14 5472 189 E H D L V G S D D L 14 5473  31 V S P K K A V A T L 13 5474  45 R S L E E E F N H F 13 5475 102 S E P Q I S R G I P 13 5476 162 Q L N P I F G E I L 13 5477 174 S I S L P A E T E L 13 5478 181 T E L T V A V F E H 13 5479 201 E T H I D L E N R F 13 5480   3 D P G D S D G V N L 12 5481   4 P G D S D G V N L I 12 5482  74 G E E E G S G H L V 12 5483  94 P E S E A V L F S E 12 5484  97 E A V L F S E P Q I 12 5485 101 F S E P Q I S R G I 12 5486 150 E R Q D T K E R Y I 12 5487 155 K E R Y I P K Q L N 12 5488 161 K Q L N P I F G E I 12 5489 206 L E N R F Y S H H R 12 5490 215 R A N C G L A S Q Y 12 5491 218 C G L A S Q Y E V W 12 5492 part 3: MHC Class I decamer analysis of 158P3D2 v.3, (aa 94-103-112, LPTEREVSVRRRSGPFALE). HLA-A*0203 decamers Pos 1 2 3 4 5 6 7 8 9 0 score Seq. ID. NO 101 S V R R R S G P F A 10 5493 102 V R R R S G P F A L  9 5494 HLA-A1 decamers Pos 1 2 3 4 5 6 7 8 9 0 score Seq. ID. NO  95 P T E R E V S V R R 16 5495  97 E R E V S V R R R S 11 5496 HLA-A26 decamers Pos 1 2 3 4 5 6 7 8 9 0 score Seq. ID. NO  99 E V S V R R R S G P 18 5497 HLA-A3 decamers Pos 1 2 3 4 5 6 7 8 9 0 score Seq. ID. NO 101 S V R R R S G P F A 20 5498  99 E V S V R R R S G P 14 5499 HLA-B*0702 decamers Pos 1 2 3 4 5 6 7 8 9 0 score Seq. ID. NO 102 V R R R S G P F A L 13 5500  94 L P T E R E V S V R 12 5501 HLA-B*4402 decamers Pos 1 2 3 4 5 6 7 8 9 0 score Seq. ID. NO 102 V R R R S G P F A L 14 5502  96 T E R E V S V R R R 12 5503  98 R E V S V R R R S G 12 5504 100 V S V R R R S G P F 11 5505 part 4: MHC Class I decamer analysis of 158P3D2 v.4 (aa 93-102-111, YLPTEREVSIWRRSGPFAL). HLA-A*0201 decamers Pos 1 2 3 4 5 6 7 8 9 0 score Seq. ID. NO 101 S I W R R S G P F A 16 5506 HLA-A*0203 decamers Pos 1 2 3 4 5 6 7 8 9 0 score Seq. ID. NO 101 S I W R R S G P F A 10 5507 102 I W R R S G P F A L  9 5508 HLA-A1 decamers Pos 1 2 3 4 5 6 7 8 9 0 score Seq. ID. NO  95 P T E R E V S I W R 20 5509  97 E R E V S I W R R S 10 5510 HLA-A26 decamers Pos 1 2 3 4 5 6 7 8 9 0 score Seq. ID. NO  99 E V S I W R R S G P 18 5511 HLA-B*0702 decamers Pos 1 2 3 4 5 6 7 8 9 0 score Seq. ID. NO 102 I W R R S G P F A L 14 5512 HLA-B*4402 decamers Pos 1 2 3 4 5 6 7 8 9 0 score Seq. ID. NO 102 I W R R S G P F A L 14 5513  95 T E R E V S I W R R 13 5514 100 V S I W R R S G P F 13 5515  98 R E V S I W R R S G 12 5516  94 L P T E R E V S I W 11 5517 part 5: MHC Class I decamer analysis of 158P3D2 v.5a (aa 121-178, VLVLQVWDYT ASLPMTSLDP WSCSYQTWCV GPGAPSSALC SWPAMGPGRG AICFAAAA). HLA-A*0201 decamers Pos 1 2 3 4 5 6 7 8 9 0 score Seq. ID. NO   9 Y T A S L P M T S L 20 5518   4 L Q V W D Y T A S L 16 5519  12 S L P M T S L D P W 16 5520   1 V L V L Q V W D Y T 15 5521   2 L V L Q V W D Y T A 15 5522  17 S L D P W S C S Y Q 14 5523  30 V G P G A P S S A L 14 5524  44 A M G P G R G A I C 14 5525  38 A L C S W P A M G P 13 5526  43 P A M G P G R G A I 13 5527   3 V L Q V W D Y T A S 12 5528  29 C V G P G A P S S A 12 5529  33 G A P S S A L C S W 11 5530   7 W D Y T A S L P M T 10 5531  21 W S C S Y Q T W C V 10 5532  36 S S A L C S W P A M 10 5533  37 S A L C S W P A M G 10 5534 HLA-A*0203 decamers Pos 1 2 3 4 5 6 7 8 9 0 score Seq. ID. NO  48 G R G A I C F A A A 27 5535  49 R G A I C F A A A A 27 5536  47 P G R G A I C F A A 19 5537 HLA-A1 decamers Pos 1 2 3 4 5 6 7 8 9 0 score Seq. ID. NO  16 T S L D P W S C S Y 19 5538   6 V W D Y T A S L P M 17 5539  17 S L D P W S C S Y Q 17 5540  11 A S L P M T S L D P 15 5541  32 P G A P S S A L C S 10 5542  40 C S W P A M G P G R  9 5543 HLA-A26 decamers Pos 1 2 3 4 5 6 7 8 9 0 score Seq. ID. NO   9 Y T A S L P M T S L 25 5544  29 C V G P G A P S S A 14 5545   3 V L Q V W D Y T A S 13 5546  12 S L P M T S L D P W 13 5547  30 V G P G A P S S A L 13 5548  45 M G P G R G A I C F 13 5549   5 Q V W D Y T A S L P 12 5550  16 T S L D P W S C S Y 12 5551  17 S L D P W S C S Y Q 12 5552  19 D P W S C S Y Q T W 12 5553 HLA-A3 decamers Pos 1 2 3 4 5 6 7 8 9 0 score Seq. ID. NO   5 Q V W D Y T A S L P 19 5554  29 C V G P G A P S S A 19 5555  17 S L D P W S C S Y Q 17 5556  38 A L C S W P A M G P 17 5557   2 L V L Q V W D Y T A 16 5558  49 R G A I C F A A A A 13 5559   3 V L Q V W D Y T A S 12 5560  44 A M G P G R G A I C 12 5561   1 V L V L Q V W D Y T 11 5562  11 A S L P M T S L D P 11 5563  12 S L P M T S L D P W 11 5564  16 T S L D P W S C S Y 11 5565  32 P G A P S S A L C S 10 5566  40 C S W P A M G P G R 10 5567  45 M G P G R G A I C F  9 5568 HLA-B*0702 decamers Pos 1 2 3 4 5 6 7 8 9 0 score Seq. ID. NO  46 G P G R G A I C F A 18 5569  42 W P A M G P G R G A 17 5570  34 A P S S A L C S W P 14 5571  30 V G P G A P S S A L 13 5572  31 G P G A P S S A L C 13 5573   4 L Q V W D Y T A S L 12 5574   9 Y T A S L P M T S L 12 5575  13 L P M T S L D P W S 12 5576  43 P A M G P G R G A I 11 5577  47 P G R G A I C F A A 11 5578  48 G R G A I C F A A A 11 5579   6 V W D Y T A S L P M 10 5580  19 D P W S C S Y Q T W 10 5581  35 P S S A L C S W P A 10 5582  49 R G A I C F A A A A 10 5583  36 S S A L C S W P A M  9 5584 HLA-B*4402 decamers Pos 1 2 3 4 5 6 7 8 9 0 score Seq. ID. NO  30 V G P G A P S S A L 14 5585  45 M G P G R G A I C F 14 5586  12 S L P M T S L D P W 13 5587  43 P A M G P G R G A I 13 5588  16 T S L D P W S C S Y 12 5589  33 G A P S S A L C S W 12 5590   4 L Q V W D Y T A S L 11 5591  19 D P W S C S Y Q T W 11 5592   9 Y T A S L P M T S L 10 5593  11 A S L P M T S L D P  8 5594

TABLE XIXC MHC Class II Analysis of 158P3D2 part 1: MHC Class II 15-mer analysis of 158P3D2 v.1 (aa 1-328). Listed are scores which correlate with the ligation strength to a defined HLA type for a sequence of amino acids. The algorithms used are based on the book “MHC Ligands and Peptide Motifs” by H. G. Rammensee, J. Bachmann and S. Stevanovic. The probability of being processed and presented is given in order to in order to predict T-cell epitopes. HLA-DRB1*0101 15 - mers Pos 1 2 3 4 5 6 7 8 9 0 1 2 3 4 5 score Seq. ID NO 126 V W D Y D R I S A N D F L G S 32 5595 308 L L V F Y T I P G Q I S Q V I 32 5596 274 N W F V N P L K T F V F F I W 31 5597 296 V L L L L V L L T V F L L L V 30 5598 71 D V H F N S L T G E G N F N W 29 5599 138 L G S L E L Q L P D M V R G A 29 5600 226 G G N V Y I L T G K V E A E F 28 5601 289 R R Y W R T L V L L L L V L L 28 5602 311 F Y T I P G Q I S Q V I F R P 28 5603 100 V S V W R R S G P F A L E E A 27 5604 183 W P V V K L K E A E D V E R E 27 5605 237 E A E F E L L T V E E A E K R 27 5606 303 L T V F L L L V F Y T I P G Q 27 5607  27 E L R V V I W N T E D V V L D 26 5608 146 P D M V R G A R G P E L C S V 26 5609 173 L F R C R R L R G W W P V V K 26 5610 219 D L E F T D M G G N V Y I L T 26 5611 292 W R T L V L L L L V L L T V F 26 5612 297 L L L L V L L T V F L L L V F 26 5613  40 L D D E N P L T G E M S S D I 25 5614 135 N D F L G S L E L Q L P D M V 25 5615 180 R G W W P V V K L K E A E D V 25 5616 294 T L V L L L L V L L T V F L L 25 5617   3 I D I F P Q D V P A P P P V D 24 5618  52 S D I Y V K S W V K G L E H D 24 5619  88 V F R F D Y L P T E R E V S V 24 5620  99 E V S V W R R S G P F A L E E 24 5621 132 I S A N D F L G S L E L Q L P 24 5622 295 L V L L L L V L L T V F L L L 24 5623 304 T V F L L L V F Y T I P G Q I 24 5624  43 E N P L T G E M S S D I Y V K 23 5625   2 W I D I F P Q D V P A P P P V 22 5626   4 D I F P Q D V P A P P P V D I 22 5627   7 P Q D V P A P P P V D I K P R 22 5628  12 A P P P V D I K P R Q P I S Y 22 5629 112 E E A E F R Q P A V L V L Q V 22 5630 151 G A R G P E L C S V Q L A R N 22 5631 225 M G G N V Y I L T G K V E A E 22 5632 299 L L V L L T V F L L L V F Y T 22 5633 307 L L L V F Y T I P G Q I S Q V 22 5634 285 F F I W R R Y W R T L V L L L 21 5635  84 N W R F V F R F D Y L P T E R 20 5636 106 S G P F A L E E A E F R Q P A 20 5637 113 E A E F R Q P A V L V L Q V W 20 5638 144 Q L P D M V R G A R G P E L C 20 5639 227 G N V Y I L T G K V E A E F E 20 5640 273 F N W F V N P L K T F V F F I 20 5641  13 P P P V D I K P R Q P I S Y E 19 5642  57 K S W V K G L E H D K Q E T D 19 5643  80 E G N F N W R F V F R F D Y L 19 5644  82 N F N W R F V F R F D Y L P T 19 5645  90 R F D Y L P T E R E V S V W R 19 5646 156 E L C S V Q L A R N G A G P R 19 5647 182 W W P V V K L K E A E D V E R 19 5648 240 F E L L T V E E A E K R P V G 19 5649 272 S F N W F V N P L K T F V F F 19 5650  35 T E D V V L D D E N P L T G E 18 5651  97 E R E V S V W R R S G P F A L 18 5652 108 P F A L E E A E F R Q P A V L 18 5653 129 Y D R I S A N D F L G S L E L 18 5654 134 A N D F L G S L E L Q L P D M 18 5655 158 C S V Q L A R N G A G P R C N 18 5656 190 E A E D V E R E A Q E A Q A G 18 5657 209 K Q R R R K G R P E D L E F T 18 5658 214 K G R P E D L E F T D M G G N 18 5659 242 L L T V E E A E K R P V G K G 18 5660 288 W R R Y W R T L V L L L L V L 18 5661  29 R V V I W N T E D V V L D D E 17 5662  34 N T E D V V L D D E N P L T G 17 5663  47 T G E M S S D I Y V K S W V K 17 5664 105 R S G P F A L E E A E F R Q P 17 5665 159 S V Q L A R N G A G P R C N L 17 5666 179 L R G W W P V V K L K E A E D 17 5667 229 V Y I L T G K V E A E F E L L 17 5668 230 Y I L T G K V E A E F E L L T 17 5669 284 V F F I W R R Y W R T L V L L 17 5670 293 R T L V L L L L V L L T V F L 17 5671 298 L L L V L L T V F L L L V F Y 17 5672 300 L V L L T V F L L L V F Y T I 17 5673 302 L L T V F L L L V F Y T I P G 17 5674  17 D I K P R Q P I S Y E L R V V 16 5675  21 R Q P I S Y E L R V V I W N T 16 5676  25 S Y E L R V V I W N T E D V V 16 5677  28 L R V V I W N T E D V V L D D 16 5678  91 F D Y L P T E R E V S V W R R 16 5679 118 Q P A V L V L Q V W D Y D R I 16 5680 119 P A V L V L Q V W D Y D R I S 16 5681 121 V L V L Q V W D Y D R I S A N 16 5682 123 V L Q V W D Y D R I S A N D F 16 5683 137 F L G S L E L Q L P D M V R G 16 5684 176 C R R L R G W W P V V K L K E 16 5685 196 R E A Q E A Q A G K K K R K Q 16 5686 218 E D L E F T D M G G N V Y I L 16 5687 261 E P L E K P S R P K T S F N W 16 5688 281 K T F V F F I W R R Y W R T L 16 5689 286 F I W R R Y W R T L V L L L L 16 5690 290 R Y W R T L V L L L L V L L T 16 5691 291 Y W R T L V L L L L V L L T V 16 5692 HLA-DRB1*0301 (DR17) 15 - mers Pos 1 2 3 4 5 6 7 8 9 0 1 2 3 4 5 score Seq. ID NO  35 T E D V V L D D E N P L T G E 37 5693  36 E D V V L D D E N P L T G E M 30 5694  60 V K G L E H D K Q E T D V H F 27 5695 134 A N D F L G S L E L Q L P D M 26 5696 229 V Y I L T G K V E A E F E L L 26 5697  47 T G E M S S D I Y V K S W V K 23 5698 292 W R T L V L L L L V L L T V F 23 5699 298 L L L V L L T V F L L L V F Y 23 5700 295 L V L L L L V L L T V F L L L 22 5701 296 V L L L L V L L T V F L L L V 22 5702 297 L L L L V L L T V F L L L V F 21 5703 300 L V L L T V F L L L V F Y T I 21 5704  15 P V D I K P R Q P I S Y E L R 20 5705  29 R V V I W N T E D V V L D D E 20 5706 118 Q P A V L V L Q V W D Y D R I 20 5707 239 E F E L L T V E E A E K R P V 20 5708 274 N W F V N P L K T F V F F I W 20 5709   3 I D I F P Q D V P A P P P V D 19 5710  53 D I Y V K S W V K G L E H D K 19 5711  74 F N S L T G E G N F N W R F V 19 5712  86 R F V F R F D Y L P T E R E V 19 5713 146 P D M V R G A R G P E L C S V 19 5714 182 W W P V V K L K E A E D V E R 19 5715 219 D L E F T D M G G N V Y I L T 19 5716  13 P P P V D I K P R Q P I S Y E 18 5717  21 R Q P I S Y E L R V V I W N T 18 5718 113 E A E F R Q P A V L V L Q V W 18 5719 130 D R I S A N D F L G S L E L Q 18 5720 157 L C S V Q L A R N G A G P R C 18 5721 213 R K G R P E D L E F T D M G G 18 5722 233 T G K V E A E F E L L T V E E 18 5723 242 L L T V E E A E K R P V G K G 18 5724 260 P E P L E K P S R P K T S F N 18 5725 284 V F F I W R R Y W R T L V L L 18 5726 HLA-DRB1*0401 (DR4Dw4) 15 - mers Pos 1 2 3 4 5 6 7 8 9 0 1 2 3 4 5 score Seq. ID NO 270 K T S F N W F V N P L K T F V 28 5727  21 R Q P I S Y E L R V V I W N T 26 5728  36 E D V V L D D E N P L T G E M 26 5729  43 E N P L T G E M S S D I Y V K 26 5730  57 K S W V K G L E H D K Q E T D 26 5731 191 A E D V E R E A Q E A Q A G K 26 5732 296 V L L L L V L L T V F L L L V 26 5733  71 D V H F N S L T G E G N F N W 22 5734  82 N F N W R F V F R F D Y L P T 22 5735  88 V F R F D Y L P T E R E V S V 22 5736  90 R F D Y L P T E R E V S V W R 22 5737 124 L Q V W D Y D R I S A N D F L 22 5738 180 R G W W P V V K L K E A E D V 22 5739 237 E A E F E L L T V E E A E K R 22 5740 285 F F I W R R Y W R T L V L L L 22 5741 289 R R Y W R T L V L L L L V L L 22 5742 303 L T V F L L L V F Y T I P G Q 22 5743 308 L L V F Y T I P G Q I S Q V I 22 5744 309 L V F Y T I P G Q I S Q V I F 22 5745  27 E L R V V I W N T E D V V L D 20 5746  35 T E D V V L D D E N P L T G E 20 5747  47 T G E M S S D I Y V K S W V K 20 5748  60 V K G L E H D K Q E T D V H F 20 5749  74 F N S L T G E G N F N W R F V 20 5750  85 W R F V F R F D Y L P T E R E 20 5751  91 F D Y L P T E R E V S V W R R 20 5752 123 V L Q V W D Y D R I S A N D F 20 5753 146 P D M V R G A R G P E L C S V 20 5754 154 G P E L C S V Q L A R N G A G 20 5755 157 L C S V Q L A R N G A G P R C 20 5756 233 T G K V E A E F E L L T V E E 20 5757 239 E F E L L T V E E A E K R P V 20 5758 242 L L T V E E A E K R P V G K G 20 5759 260 P E P L E K P S R P K T S F N 20 5760 274 N W F V N P L K T F V F F I W 20 5761 281 K T F V F F I W R R Y W R T L 20 5762 292 W R T L V L L L L V L L T V F 20 5763 293 R T L V L L L L V L L T V F L 20 5764 294 T L V L L L L V L L T V F L L 20 5765 297 L L L L V L L T V F L L L V F 20 5766 299 L L V L L T V F L L L V F Y T 20 5767 302 L L T V F L L L V F Y T I P G 20 5768 305 V F L L L V F Y T I P G Q I S 20 5769 311 F Y T I P G Q I S Q V I F R P 20 5770  50 M S S D I Y V K S W V K G L E 18 5771  65 H D K Q E T D V H F N S L T G 18 5772 109 F A L E E A E F R Q P A V L V 18 5773 110 A L E E A E F R Q P A V L V L 18 5774 132 I S A N D F L G S L E L Q L P 18 5775 151 G A R G P E L C S V Q L A R N 18 5776 156 E L C S V Q L A R N G A G P R 18 5777 188 L K E A E D V E R E A Q E A Q 18 5778 194 V E R E A Q E A Q A G K K K R 18 5779 225 M G G N V Y I L T G K V E A E 18 5780   3 I D I F P Q D V P A P P P V D 16 5781  30 V V I W N T E D V V L D D E N 16 5782  52 S D I Y V K S W V K G L E H D 16 5783  56 V K S W V K G L E H D K Q E T 16 5784  86 R F V F R F D Y L P T E R E V 16 5785 100 V S V W R R S G P F A L E E A 16 5786 106 S G P F A L E E A E F R Q P A 16 5787 113 E A E F R Q P A V L V L Q V W 16 5788 126 V W D Y D R I S A N D F L G S 16 5789 134 A N D F L G S L E L Q L P D M 16 5790 179 L R G W W P V V K L K E A E D 16 5791 227 G N V Y I L T G K V E A E F E 16 5792 273 F N W F V N P L K T F V F F I 16 5793 280 L K T F V F F I W R R Y W R T 16 5794 282 T F V F F I W R R Y W R T L V 16 5795 288 W R R Y W R T L V L L L L V L 16 5796  13 P P P V D I K P R Q P I S Y E 15 5797   7 P Q D V P A P P P V D I K P R 14 5798  25 S Y E L R V V I W N T E D V V 14 5799  28 L R V V I W N T E D V V L D D 14 5800  29 R V V I W N T E D V V L D D E 14 5801  37 D V V L D D E N P L T G E M S 14 5802  97 E R E V S V W R R S G P F A L 14 5803 108 P F A L E E A E F R Q P A V L 14 5804 118 Q P A V L V L Q V W D Y D R I 14 5805 120 A V L V L Q V W D Y D R I S A 14 5806 121 V L V L Q V W D Y D R I S A N 14 5807 129 Y D R I S A N D F L G S L E L 14 5808 135 N D F L G S L E L Q L P D M V 14 5809 138 L G S L E L Q L P D M V R G A 14 5810 142 E L Q L P D M V R G A R G P E 14 5811 145 L P D M V R G A R G P E L C S 14 5812 170 R C N L F R C R R L R G W W P 14 5813 176 C R R L R G W W P V V K L K E 14 5814 182 W W P V V K L K E A E D V E R 14 5815 185 V V K L K E A E D V E R E A Q 14 5816 217 P E D L E F T D M G G N V Y I 14 5817 222 F T D M G G N V Y I L T G K V 14 5818 226 G G N V Y I L T G K V E A E F 14 5819 240 F E L L T V E E A E K R P V G 14 5820 277 V N P L K T F V F F I W R R Y 14 5821 295 L V L L L L V L L T V F L L L 14 5822 298 L L L V L L T V F L L L V F Y 14 5823 300 L V L L T V F L L L V F Y T I 14 5824 304 T V F L L L V F Y T I P G Q I 14 5825 306 F L L L V F Y T I P G Q I S Q 14 5826 307 L L L V F Y T I P G Q I S Q V 14 5827 HLA-DRB1*1101 15 - mers Pos 1 2 3 4 5 6 7 8 9 0 1 2 3 4 5 score Seq. ID NO 179 L R G W W P V V K L K E A E D 27 5828  90 R F D Y L P T E R E V S V W R 25 5829  82 N F N W R F V F R F D Y L P T 24 5830 227 G N V Y I L T G K V E A E F E 24 5831 170 R C N L F R C R R L R G W W P 23 5832 180 R G W W P V V K L K E A E D V 23 5833 308 L L V F Y T I P G Q I S Q V I 23 5834 142 E L Q L P D M V R G A R G P E 22 5835 237 E A E F E L L T V E E A E K R 22 5836 281 K T F V F F I W R R Y W R T L 22 5837  57 K S W V K G L E H D K Q E T D 21 5838  96 T E R E V S V W R R S G P F A 21 5839  97 E R E V S V W R R S G P F A L 21 5840 123 V L Q V W D Y D R I S A N D F 20 5841 156 E L C S V Q L A R N G A G P R 20 5842 135 N D F L G S L E L Q L P D M V 19 5843 219 D L E F T D M G G N V Y I L T 19 5844 282 T F V F F I W R R Y W R T L V 19 5845 285 F F I W R R Y W R T L V L L L 19 5846 289 R R Y W R T L V L L L L V L L 19 5847 304 T V F L L L V F Y T I P G Q I 19 5848   3 I D I F P Q D V P A P P P V D 18 5849  88 V F R F D Y L P T E R E V S V 18 5850 273 F N W F V N P L K T F V F F I 18 5851 303 L T V F L L L V F Y T I P G Q 18 5852  53 D I Y V K S W V K G L E H D K 17 5853  84 N W R F V F R F D Y L P T E R 17 5854  65 H D K Q E T D V H F N S L T G 16 5855  71 D V H F N S L T G E G N F N W 16 5856 126 V W D Y D R I S A N D F L G S 16 5857 167 A G P R C N L F R C R R L R G 16 5858 204 G K K K R K Q R R R K G R P E 16 5859 206 K K R K Q R R R K G R P E D L 16 5860 247 E A E K R P V G K G R K Q P E 16 5861  21 R Q P I S Y E L R V V I W N T 15 5862 242 L L T V E E A E K R P V G K G 15 5863 243 L T V E E A E K R P V G K G R 15 5864 260 P E P L E K P S R P K T S F N 15 5865  13 P P P V D I K P R Q P I S Y E 14 5866  51 S S D I Y V K S W V K G L E H 14 5867 109 F A L E E A E F R Q P A V L V 14 5868 140 S L E L Q L P D M V R G A R G 14 5869 143 L Q L P D M V R G A R G P E L 14 5870 145 L P D M V R G A R G P E L C S 14 5871 154 G P E L C S V Q L A R N G A G 14 5872 188 L K E A E D V E R E A Q E A Q 14 5873 249 E K R P V G K G R K Q P E P L 14 5874 250 K R P V G K G R K Q P E P L E 14 5875 257 R K Q P E P L E K P S R P K T 14 5876 294 T L V L L L L V L L T V F L L 14 5877   2 W I D I F P Q D V P A P P P V 13 5878  12 A P P P V D I K P R Q P I S Y 13 5879  25 S Y E L R V V I W N T E D V V 13 5880  34 N T E D V V L D D E N P L T G 13 5881  47 T G E M S S D I Y V K S W V K 13 5882 108 P F A L E E A E F R Q P A V L 13 5883 118 Q P A V L V L Q V W D Y D R I 13 5884 226 G G N V Y I L T G K V E A E F 13 5885 270 K T S F N W F V N P L K T F V 13 5886 274 N W F V N P L K T F V F F I W 13 5887 280 L K T F V F F I W R R Y W R T 13 5888 292 W R T L V L L L L V L L T V F 13 5889 293 R T L V L L L L V L L T V F L 13 5890 295 L V L L L L V L L T V F L L L 13 5891 296 V L L L L V L L T V F L L L V 13 5892 297 L L L L V L L T V F L L L V F 13 5893 299 L L V L L T V F L L L V F Y T 13 5894 302 L L T V F L L L V F Y T I P G 13 5895 305 V F L L L V F Y T I P G Q I S 13 5896 part 2: MHC Class II 15-mer analysis of 158P3D2 v.2a (aa 1-236) HLA-DRB1*0101 15 - mers Pos 1 2 3 4 5 6 7 8 9 0 1 2 3 4 5 score Seq. ID NO 119 L V R V Y V V K A T N L A P A 31 5897 160 P K Q L N P I F G E I L E L S 31 5898  61 F P L Y R G Q G G Q D G G G E 27 5899  90 F L I Y P E S E A V L F S E P 27 5900  80 G H L V G K F K G S F L I Y P 26 5901 113 N R P I K L L V R V Y V V K A 26 5902 120 V R V Y V V K A T N L A P A D 25 5903 139 A D P Y V V V S A G R E R Q D 25 5904  96 S E A V L F S E P Q I S R G I 24 5905 116 I K L L V R V Y V V K A T N L 24 5906 156 E R Y I P K Q L N P I F G E I 24 5907 164 N P I F G E I L E L S I S L P 24 5908 167 F G E I L E L S I S L P A E T 24 5909  16 V G E I Q D Q G E A E V K G T 23 5910  21 D Q G E A E V K G T V S P K K 23 5911  88 G S F L I Y P E S E A V L F S 23 5912  99 V L F S E P Q I S R G I P Q N 23 5913 107 S R G I P Q N R P I K L L V R 23 5914 124 V V K A T N L A P A D P N G K 23 5915 168 G E I L E L S I S L P A E T E 23 5916   9 G V N L I S M V G E I Q D Q G 22 5917  25 A E V K G T V S P K K A V A T 22 5918  31 V S P K K A V A T L K I Y N R 22 5919  54 F E D W L N V F P L Y R G Q G 22 5920 187 V F E H D L V G S D D L I G E 22 5921 217 N C G L A S Q Y E V W V Q Q G 22 5922  58 L N V F P L Y R G Q G G Q D G 21 5923  87 K G S F L I Y P E S E A V L F 21 5924 165 P I F G E I L E L S I S L P A 20 5925 221 A S Q Y E V W V Q Q G P Q E P 20 5926  40 L K I Y N R S L E E E F N H F 19 5927  83 V G K F K G S F L I Y P E S E 19 5928 121 R V Y V V K A T N L A P A D P 19 5929   7 S D G V N L I S M V G E I Q D 18 5930  51 F N H F E D W L N V F P L Y R 18 5931 140 D P Y V V V S A G R E R Q D T 18 5932 155 K E R Y I P K Q L N P I F G E 18 5933 172 E L S I S L P A E T E L T V A 18 5934 184 T V A V F E H D L V G S D D L 18 5935 208 N R F Y S H H R A N C G L A S 18 5936 211 Y S H H R A N C G L A S Q Y E 18 5937  14 S M V G E I Q D Q C E A E V K 17 5938  36 A V A T L K I Y N R S L E E E 17 5939  76 E E G S G H L V G K F K G S F 17 5940  79 S G H L V G K F K G S F L I Y 17 5941  81 H L V G K F K G S F L I Y P E 17 5942  85 K F K G S F L I Y P E S E A V 17 5943 122 V Y V V K A T N L A P A D P N 17 5944 170 I L E L S I S L P A E T E L T 17 5945 177 L P A E T E L T V A V F E H D 17 5946 182 E L T V A V F E H D L V G S D 17 5947 193 V G S D D L I G E T H I D L E 17 5948 201 E T H I D L E N R F Y S H H R 17 5949   1 M D D P G D S D G V N L I S M 16 5950  10 V N L I S M V G E I Q D Q G E 16 5951  13 I S M V G E I Q D Q G E A E V 16 5952  24 E A E V K G T V S P K K A V A 16 5953  28 K G T V S P K K A V A T L K I 16 5954  34 K K A V A T L K I Y N R S L E 16 5955  52 N H F E D W L N V F P L Y R G 16 5956  57 W L N V F P L Y R G Q G G Q D 16 5957  60 V F P L Y R G Q G G Q D G G G 16 5958  64 Y R G Q G G Q D G G G E E E G 16 5959  72 G G G E E E G S G H L V G K F 16 5960  89 S F L I Y P E S E A V L F S E 16 5961 104 P Q I S R G I P Q N R P I K L 16 5962 127 A T N L A P A D P N G K A D P 16 5963 133 A D P N G K A D P Y V V V S A 16 5964 163 L N P I F G E I L E L S I S L 16 5965 171 L E L S I S L P A E T E L T V 16 5966 174 S I S L P A E T E L T V A V F 16 5967 186 A V F E H D L V G S D D L I G 16 5968 192 L V G S D D L I G E T H I D L 16 5969 195 S D D L I G E T H I D L E N R 16 5970   6 D S D G V N L I S M V G E I Q 15 5971  22 Q G E A E V K G T V S P K K A 15 5972  71 D G G G E E E G S G H L V G K 15 5973 114 R P I K L L V R V Y V V K A T 15 5974 152 Q D T K E R Y I P K Q L N P I 15 5975 157 R Y I P K Q L N P I F G E I L 15 5976 166 I F G E I L E L S I S L P A E 15 5977 181 T E L T V A V F E H D L V G S 15 5978 HLA-DRB1*0301 (DR17) 15 - mers Pos 1 2 3 4 5 6 7 8 9 0 1 2 3 4 5 score Seq. ID NO  89 S F L I Y P E S E A V L F S E 27 5979 199 I G E T H I D L E N R F Y S H 26 5980  79 S G H L V G K F K G S F L I Y 25 5981 147 A G R E R Q D T K E R Y I P K 25 5982 156 E R Y I P K Q L N P I F G E I 25 5983 172 E L S I S L P A E T E L T V A 25 5984 190 H D L V G S D D L I G E T H I 21 5985  50 E F N H F E D W L N V F P L Y 20 5986 119 L V R V Y V V K A T N L A P A 20 5987  80 G H L V G K F K G S F L I Y P 19 5988 107 S R G I P Q N R P I K L L V R 19 5989 113 N R P I K L L V R V Y V V K A 19 5990 121 R V Y V V K A T N L A P A D P 19 5991 141 P Y V V V S A G R E R Q D T K 19 5992 160 P K Q L N P I F G E I L E L S 19 5993 185 V A V F E H D L V G S D D L I 19 5994 195 S D D L I G E T H I D L E N R 19 5995  10 V N L I S M V G E I Q D Q G E 18 5996  16 V G E I Q D Q G E A E V K G T 18 5997  37 V A T L K I Y N R S L E E E F 18 5998  97 E A V L F S E P Q I S R G I P 18 5999 128 T N L A P A D P N G K A D P Y 18 6000 217 N C G L A S Q Y E V W V Q Q G 18 6001  12 L I S M V G E I Q D Q G E A E 17 6002  44 N R S L E E E F N H F E D W L 17 6003  57 W L N V F P L Y R G Q G G Q D 17 6004  87 K G S F L I Y P E S E A V L F 17 6005 164 N P I F G E I L E L S I S L P 17 6006 174 S I S L P A E T E L T V A V F 17 6007 201 E T H I D L E N R F Y S H H R 17 6008 207 E N R F Y S H H R A N C G L A 17 6009  36 A V A T L K I Y N R S L E E E 16 6010  51 F N H F E D W L N V F P L Y R 16 6011 142 Y V V V S A G R E R Q D T K E 16 6012 200 G E T H I D L E N R F Y S H H 16 6013  40 L K I Y N R S L E E E F N H F 15 6014 167 F G E I L E L S I S L P A E T 15 6015 181 T E L T V A V F E H D L V G S 15 6016   2 D D P G D S D G V N L I S M V 14 6017  28 K G T V S P K K A V A T L K I 14 6018  47 L E E E F N H F E D W L N V F 14 6019  96 S E A V L F S E P Q I S R G I 14 6020 209 R F Y S H H R A N C G L A S Q 14 6021   7 S D G V N L I S M V G E I Q D 13 6022  43 Y N R S L E E E F N H F E D W 13 6023  88 G S F L I Y P E S E A V L F S 13 6024 115 P I K L L V R V Y V V K A T N 13 6025 116 I K L L V R V Y V V K A T N L 13 6026 134 D P N G K A D P Y V V V S A G 13 6027 HLA-DRB1*0401 (DR4Dw4) 15 - mers Pos 1 2 3 4 5 6 7 8 9 0 1 2 3 4 5 score Seq. ID NO 113 N R P I K L L V R V Y V V K A 26 6028 141 P Y V V V S A G R E R Q D T K 26 6029 182 E L T V A V F E H D L V G S D 26 6030 195 S D D L I G E T H I D L E N R 26 6031 201 E T H I D L E N R F Y S H H R 26 6032  48 E E E F N H F E D W L N V F P 22 6033 164 N P I F G E I L E L S I S L P 22 6034  12 L I S M V G E I Q D Q G E A E 20 6035  24 E A E V K G T V S P K K A V A 20 6036  44 N R S L E E E F N H F E D W L 20 6037  57 W L N V F P L Y R G Q G G Q D 20 6038  80 G H L V G K F K G S F L I Y P 20 6039  88 G S F L I Y P E S E A V L F S 20 6040  89 S F L I Y P E S E A V L F S E 20 6041  97 E A V L F S E P Q I S R G I P 20 6042 116 I K L L V R V Y V V K A T N L 20 6043 119 L V R V Y V V K A T N L A P A 20 6044 121 R V Y V V K A T N L A P A D P 20 6045 127 A T N L A P A D P N G K A D P 20 6046 160 P K Q L N P I F G E I L E L S 20 6047 163 L N P I F G E I L E L S I S L 20 6048 168 G E I L E L S I S L P A E T E 20 6049 174 S I S L P A E T E L T V A V F 20 6050  31 V S P K K A V A T L K I Y N R 18 6051  36 A V A T L K I Y N R S L E E E 18 6052  71 D G G G E E E G S G H L V G K 18 6053  94 P E S E A V L F S E P Q I S R 18 6054 128 T N L A P A D P N G K A D P Y 18 6055 144 V V S A G R E R Q D T K E R Y 18 6056 166 I F G E I L E L S I S L P A E 18 6057 173 L S I S L P A E T E L T V A V 18 6058 176 S L P A E T E L T V A V F E H 18 6059 187 V F E H D L V G S D D L I G E 18 6060 215 R A N C G L A S Q Y E V W V Q 18 6061 222 S Q Y E V W V Q Q G P Q E P F 18 6062 120 V R V Y V V K A T N L A P A D 17 6063  51 F N H F E D W L N V F P L Y R 16 6064  54 F E D W L N V F P L Y R G Q G 16 6065  87 K G S F L I Y P E S E A V L F 16 6066 139 A D P Y V V V S A G R E R Q D 16 6067 185 V A V F E H D L V G S D D L I 16 6068 207 E N R F Y S H H R A N C G L A 16 6069 221 A S Q Y E V W V Q Q G P Q E P 16 6070   7 S D G V N L I S M V G E I Q D 14 6071   9 G V N L I S M V G E I Q D Q G 14 6072  10 V N L I S M V G E I Q D Q G E 14 6073  13 I S M V G E I Q D Q G E A E V 14 6074  16 V G E I Q D Q G E A E V K G T 14 6075  34 K K A V A T L K I Y N R S L E 14 6076  37 V A T L K I Y N R S L E E E F 14 6077  55 E D W L N V F P L Y R G Q G G 14 6078  96 S E A V L F S E P Q I S R G I 14 6079 107 S R G I P Q N R P I K L L V R 14 6080 117 K L L V R V Y V V K A T N L A 14 6081 122 V Y V V K A T N L A P A D P N 14 6082 156 E R Y I P K Q L N P I F G E I 14 6083 167 F G E I L E L S I S L P A E T 14 6084 170 I L E L S I S L P A E T E L T 14 6085 172 E L S I S L P A E T E L T V A 14 6086 180 E T E L T V A V F E H D L V G 14 6087 184 T V A V F E H D L V G S D D L 14 6088 190 H D L V G S D D L I G E T H I 14 6089 217 N C G L A S Q Y E V W V Q Q G 14 6090 HLA-DRB1*1101 15 - mers Pos 1 2 3 4 5 6 7 8 9 0 1 2 3 4 5 score Seq. ID NO  57 W L N V F P L Y R G Q G G Q D 27 6091 113 N R P I K L L V R V Y V V K A 22 6092  77 E G S G H L V G K F K G S F L 21 6093 100 L F S E P Q I S R G I P Q N R 21 6094 201 E T H I D L E N R F Y S H H R 20 6095 116 I K L L V R V Y V V K A T N L 19 6096 139 A D P Y V V V S A G R E R Q D 19 6097 167 F G E I L E L S I S L P A E T 19 6098 221 A S Q Y E V W V Q Q G P Q E P 19 6099  98 A V L F S E P Q I S R G I P Q 18 6100 120 V R V Y V V K A T N L A P A D 18 6101 207 E N R F Y S H H R A N C G L A 18 6102  51 F N H F E D W L N V F P L Y R 16 6103  54 F E D W L N V F P L Y R G Q G 16 6104  58 L N V F P L Y R G Q G G Q D G 16 6105  61 F P L Y R G Q G G Q D G G G E 16 6106  83 V G K F K G S F L I Y P E S E 16 6107  87 K G S F L I Y P E S E A V L F 16 6108 118 L L V R V Y V V K A T N L A P 16 6109 141 P Y V V V S A G R E R Q D T K 16 6110 164 N P I F G E I L E L S I S L P 16 6111 182 E L T V A V F E H D L V G S D 16 6112 205 D L E N R F Y S H H R A N C G 16 6113 208 N R F Y S H H R A N C G L A S 16 6114   9 G V N L I S M V G E I Q D Q G 15 6115  27 V K G T V S P K K A V A T L K 15 6116  37 V A T L K I Y N R S L E E E F 15 6117  55 E D W L N V F P L Y R G Q G G 15 6118  73 G G E E E G S G H L V G K F K 15 6119 149 R E R Q D T K E R Y I P K Q L 15 6120 153 D T K E R Y I P K Q L N P I F 15 6121  79 S G H L V G K F K G S F L I Y 14 6122 104 P Q I S R G I P Q N R P I K L 14 6123 124 V V K A T N L A P A D P N G K 14 6124 130 L A P A D P N G K A D P Y V V 14 6125 137 G K A D P Y V V V S A G R E R 14 6126 163 L N P I F G E I L E L S I S L 14 6127 195 S D D L I G E T H I D L E N R 14 6128   6 D S D G V N L I S M V G E I Q 13 6129  21 D Q G E A E V K G T V S P K K 13 6130  25 A E V K G T V S P K K A V A T 13 6131  96 S E A V L F S E P Q I S R G I 13 6132 119 L V R V Y V V K A T N L A P A 13 6133 160 P K Q L N P I F G E I L E L S 13 6134 165 P I F G E I L E L S I S L P A 13 6135 184 T V A V F E H D L V G S D D L 13 6136 189 E H D L V G S D D L I G E T H 13 6137 part 3: MHC Class II 15-mer analysis of 158P3D2 v.3 (aa 89-103-117, FRFDYLPTEREVSVRRRSGPFALEEAEFR) HLA-DRB1*0101 15 - mers Pos 1 2 3 4 5 6 7 8 9 0 1 2 3 4 5 score Seq. ID NO  11 E V S V R R R S G P F A L E E 26 6138   2 R F D Y L P T E R E V S V R R 19 6139   9 E R E V S V R R R S G P F A L 18 6140  12 V S V R R R S G P F A L E E A 17 6141   3 F D Y L P T E R E V S V R R R 16 6142  10 R E V S V R R R S G P F A L E 15 6143 HLA-DRB1*0301 (DR17) 15 - mers Pos 1 2 3 4 5 6 7 8 9 0 1 2 3 4 5 score Seq. ID NO   3 F D Y L P T E R E V S V R R R 17 6144   9 E R E V S V R R R S G P F A L 16 6145  11 E V S V R R R S G P F A L E E 12 6146  12 V S V R R R S G P F A L E E A 11 6147   2 R F D Y L P T E R E V S V R R 10 6148  10 R E V S V R R R S G P F A L E  9 6149   8 T E R E V S V R R R S G P F A  8 6150 HLA-DRB1*0401 (DR4Dw4) 15 - mers Pos 1 2 3 4 5 6 7 8 9 0 1 2 3 4 5 score Seq. ID NO   2 R F D Y L P T E R E V S V R R 22 6151   3 F D Y L P T E R E V S V R R R 20 6152   5 Y L P T E R E V S V R R R S G 12 6153   7 P T E R E V S V R R R S G P F 12 6154   8 T E R E V S V R R R S G P F A 12 6155  15 R R R S G P F A L E E A E F R 12 6156 HLA-DRB1*1101 15 - mers Pos 1 2 3 4 5 6 7 8 9 0 1 2 3 4 5 score Seq. ID NO   2 R F D Y L P T E R E V S V R R 25 6157   8 T E R E V S V R R R S G P F A 21 6158   9 E R E V S V R R R S G P F A L 21 6159   7 P T E R E V S V R R R S G P F 20 6160 11 E V S V R R R S G P F A L E E 12 6161 part 4: MHC Class II 15-mer analysis of 158P3D2 v. 4 (aa 88-102-116, VFRFDYLPTEREVSIWRRSGPFALEEAEF) HLA-DRB1*0101 15 - mers Pos 1 2 3 4 5 6 7 8 9 0 1 2 3 4 5 score Seq. ID NO  13 V S I W R R S G P F A L E E A 27 6162   1 V F R F D Y L P T E R E V S I 24 6163  12 E V S I W R R S G P F A L E E 24 6164  10 E R E V S I W R R S G P F A L 21 6165   3 R F D Y L P T E R E V S I W R 19 6166   4 F D Y L P T E R E V S I W R R 16 6167  11 R E V S I W R R S G P F A L E 14 6168 HLA-DRB1*0301 (DR17) 15 - mers Pos 1 2 3 4 5 6 7 8 9 0 1 2 3 4 5 score Seq. ID NO   4 F D Y L P T E R E V S I W R R 17 6169  10 E R E V S I W R R S G P F A L 16 6170  12 E V S I W R R S G P F A L E E 11 6171  13 V S I W R R S G P F A L E E A 11 6172   3 R F D Y L P T E R E V S I W R 10 6173   1 V F R F D Y L P T E R E V S I  9 6174  11 R E V S I W R R S G P F A L E  9 6175   9 T E R E V S I W R R S G P F A  8 6176 HLA-DRB1*0401 (DR4Dw4) 15 - mers Pos 1 2 3 4 5 6 7 8 9 0 1 2 3 4 5 score Seq. ID NO   1 V F R F D Y L P T E R E V S I 22 6177   3 R F D Y L P T E R E V S I W R 22 6178   4 F D Y L P T E R E V S I W R R 20 6179  13 V S I W R R S G P F A L E E A 16 6180  10 E R E V S I W R R S G P F A L 14 6181   6 Y L P T E R E V S I W R R S G 12 6182   9 T E R E V S I W R R S G P F A 12 6183 HLA-DRB1*1101 15 - mers Pos 1 2 3 4 5 6 7 8 9 0 1 2 3 4 5 score Seq. ID NO   3 R F D Y L P T E R E V S I W R 25 6184   9 T E R E V S I W R R S G P F A 21 6185  10 E R E V S I W R R S G P F A L 20 6186   1 V F R F D Y L P T E R E V S I 18 6187  12 E V S I W R R S G P F A L E E 12 6188 part 5: MHC Class II 15-mer analysis of 158P3D2 v.5a (aa 116-178). HLA-DRB1*0101 15 - mers Pos 1 2 3 4 5 6 7 8 9 0 1 2 3 4 5 score Seq. ID NO  30 Y Q T W C V G P G A P S S A L 26 6189  41 S S A L C S W P A M G P G R G 26 6190  11 V W D Y T A S L P M T S L D P 25 6191   8 V L Q V W D Y T A S L P M T S 24 6192   9 L Q V W D Y T A S L P M T S L 24 6193  17 S L P M T S L D P W S C S Y Q 23 6194  44 L C S W P A M G P G R G A I C 23 6195   5 A V L V L Q V W D Y T A S L P 22 6196  32 T W C V G P G A P S S A L C S 22 6197  38 G A P S S A L C S W P A M G P 22 6198  29 S Y Q T W C V G P G A P S S A 21 6199  14 Y T A S L P M T S L D P W S C 20 6200  45 C S W P A M G P G R G A I C F 20 6201  28 C S Y Q T W C V G P G A P S S 19 6202  31 Q T W C V G P G A P S S A L C 18 6203   6 V L V L Q V W D Y T A S L P M 17 6204  12 W D Y T A S L P M T S L D P W 17 6205  48 P A M G P G R G A I C F A A A 17 6206   3 Q P A V L V L Q V W D Y T A S 16 6207   4 P A V L V L Q V W D Y T A S L 16 6208  35 V G P G A P S S A L C S W P A 16 6209  47 W P A M G P G R G A I C F A A 16 6210   7 L V L Q V W D Y T A S L P M T 15 6211  24 D P W S C S Y Q T W C V G P G 14 6212  33 W C V G P G A P S S A L C S W 14 6213 HLA-DRB1*0301 (DR17) 15 - mers Pos 1 2 3 4 5 6 7 8 9 0 1 2 3 4 5 score Seq. ID NO   3 Q P A V L V L Q V W D Y T A S 20 6214   7 L V L Q V W D Y T A S L P M T 20 6215   4 P A V L V L Q V W D Y T A S L 12 6216   5 A V L V L Q V W D Y T A S L P 12 6217  15 T A S L P M T S L D P W S C S 12 6218  17 S L P M T S L D P W S C S Y Q 12 6219  18 L P M T S L D P W S C S Y Q T 12 6220  20 M T S L D P W S C S Y Q T W C 12 6221  41 S S A L C S W P A M G P G R G 12 6222  47 W P A M G P G R G A I C F A A 12 6223   6 V L V L Q V W D Y T A S L P M 11 6224   8 V L Q V W D Y T A S L P M T S 11 6225  32 T W C V G P G A P S S A L C S 11 6226   2 R Q P A V L V L Q V W D Y T A 10 6227  12 W D Y T A S L P M T S L D P W 10 6228  19 P M T S L D P W S C S Y Q T W 10 6229  33 W C V G P G A P S S A L C S W 10 6230 HLA-DRB1*0401 (DR4Dw4) 15 - mers Pos 1 2 3 4 5 6 7 8 9 0 1 2 3 4 5 score Seq. ID NO   9 L Q V W D Y T A S L P M T S L 22 6231   5 A V L V L Q V W D Y T A S L P 20 6232   7 L V L Q V W D Y T A S L P M T 18 6233  33 W C V G P G A P S S A L C S W 18 6234  38 G A P S S A L C S W P A M G P 18 6235  11 V W D Y T A S L P M T S L D P 16 6236  23 L D P W S C S Y Q T W C V G P 16 6237  30 Y Q T W C V G P G A P S S A L 16 6238   3 Q P A V L V L Q V W D Y T A S 14 6239   4 P A V L V L Q V W D Y T A S L 14 6240   6 V L V L Q V W D Y T A S L P M 14 6241  17 S L P M T S L D P W S C S Y Q 14 6242  20 M T S L D P W S C S Y Q T W C 14 6243  32 T W C V G P G A P S S A L C S 14 6244   2 R Q P A V L V L Q V W D Y T A 12 6245  10 Q V W D Y T A S L P M T S L D 12 6246  12 W D Y T A S L P M T S L D P W 12 6247  18 L P M T S L D P W S C S Y Q T 12 6248  21 T S L D P W S C S Y Q T W C V 12 6249  24 D P W S C S Y Q T W C V G P G 12 6250  46 S W P A M G P G R G A I C F A 12 6251 HLA-DRB1*1101 15 - mers Pos 1 2 3 4 5 6 7 8 9 0 1 2 3 4 5 score Seq. ID NO  44 L C S W P A M G P G R G A I C 24 6252   5 A V L V L Q V W D Y T A S L P 18 6253  11 V W D Y T A S L P M T S L D P 18 6254  30 Y Q T W C V G P G A P S S A L 17 6255  27 S C S Y Q T W C V G P G A P S 16 6256  17 S L P M T S L D P W S C S Y Q 14 6257   3 Q P A V L V L Q V W D Y T A S 13 6258   6 V L V L Q V W D Y T A S L P M 13 6259   8 V L Q V W D Y T A S L P M T S 13 6260  14 Y T A S L P M T S L D P W S C 12 6261  29 S Y Q T W C V G P G A P S S A 12 6262  32 T W C V G P G A P S S A L C S 12 6263  38 G A P S S A L C S W P A M G P 12 6264  41 S S A L C S W P A M G P G R G 12 6265

TABLE XX Frequently Occurring Motifs avrg. % Name identity Description Potential Function zf-C2H2 34% Zinc finger, C2H2 type Nucleic acid-binding protein functions as tanscription factor, nuclear location probable cytochrome_b_N 68% Cytochrome b(N- membrane bound oxidase, generate terminal)/b6/petB superoxide ig 19% Immunoglobulin domain domains are one hundred amino acids long and include a conserved intradomain disulfide bond. WD40 18% WD domain, G-beta repeat tandem repeats of about 40 residues, each containing a Trp-Asp motif. Function in signal transduction and protein interaction PDZ 23% PDZ domain may function in targeting signaling molecules to sub-membranous sites LRR 28% Leucine Rich Repeat short sequence motifs involved in protein- protein interactions pkinase 23% Protein kinase domain conserved catalytic core common to both serine/threonine and tyrosine protein kinases containing an ATP binding site and a catalytic site PH 16% PH domain pleckstrin homology involved in intracellular signaling or as constituents of the cytoskeleton EGF 34% EGF-like domain 30-40 amino-acid long found in the extracellular domain of membrane-bound proteins or in secreted proteins rvt 49% Reverse transcriptase (RNA-dependent DNA polymerase) ank 25% Ank repeat Cytoplasmic protein, associates integral membrane proteins to the cytoskeleton oxidored_q1 32% NADH- membrane associated. Involved in proton Ubiquinone/plastoquinone translocation across the membrane (complex I), various chains efhand 24% EF hand calcium-binding domain, consists of a12 residue loop flanked on both sides by a 12 residue alpha-helical domain rvp 79% Retroviral aspartyl protease Aspartyl or acid proteases, centered on a catalytic aspartyl residue Collagen 42% Collagen triple helix repeat extracellular structural proteins involved in (20 copies) formation of connective tissue. The sequence consists of the G-X-Y and the polypeptide chains forms a triple helix. fn3 20% Fibronectin type III domain Located in the extracellular ligand-binding region of receptors and is about 200 amino acid residues long with two pairs of cysteines involved in disulfide bonds 7tm_1 19% 7 transmembrane receptor seven hydrophobic transmembrane regions, (rhodopsin family) with the N-terminus located extracellularly while the C-terminus is cytoplasmic. Signal through G proteins

TABLE XXI Motifs and Post-translational Modifications of 158P3D2 Protein kinase C phosphorylation site 96-98 TeR 233-235 TgK Casein kinase II phosphorylation site. 96-99 TerE 133-136 SanD 244-247 TveE Amidation site. 203-206 aGKK 255-258 kGRK Aminoacyl-transfer RNA synthetases class-II signature .1  89-113 FRfDylpterevsvwrRsgpFaleE C2-domain.  13-142

TABLE XXII Properties of 158P3D2 Bioinformatic Program Outcome Variant 1 ORF ORF finder Protein length 328 aa Transmembrane region TM Pred 1 TM helix 295-312 HMMTop N terminus extracellular, 1TM helix aa 295-314 Sosui 1 TM helix 291-313 TMHMM N terminus extracellular, 1 TM helix 292-314 Signal Peptide Signal P none pI pI/MW tool 8.64 Molecular weight pI/MW tool 38.4 kDa Localization PSORT 85% endoplasmic reticulum, 64% peroxisome, 44% plasma membrane, 35% nucleus PSORT II 33.3% vesicles of secretory system, 22.2% cytoplasmic Motifs Pfam 7TM chemoreceptor Prints No significant motif Blocks C2 domain Variant 2A ORF ORF finder Protein length 236 aa Transmembrane region TM Pred no TM HMMTop no TM, extracellular Sosui no TM, soluble protein TMHMM no TM Signal Peptide Signal P none pI pI/MW tool 4.7 Molecular weight pI/MW tool 26.1 kDa Localization PSORT 65% cytoplasm, 10% mitochondrial matrix space, 10% lysosome PSORT II 60.9% cytoplasm, 21.7% nuclear Motifs Pfam C2 domain, glutamine synthetase Prints no significant motif Blocks C2 domain Variant 2B ORF ORF finder Protein length 181 aa Transmembrane region TM Pred 1TM helix at aa 148-165 HMMTop N terminus intracellular 1TM helix at aa 148-167 Sosui 1TM helix at aa 144-166 TMHMM N terminus intracellular 1TM helix at aa 148-167 Signal Peptide Signal P none pI pI/MW tool 10.37 Molecular weight pI/MW tool 21.19 kDa Localization PSORT 85% endoplasmic reticulum, 58% peroxisome, 44% plasma membrane PSORT II 33.3% vesicles of secretory system, 22.2% plasma membrane Motifs Pfam 7TM chemoreceptor Prints No significant motif Blocks no significant motif Variant 5A ORF ORF finder Protein length 178 aa Transmembrane region TM Pred N terminus extracellular, 1 TM helix 145-165 HMMTop N terminus extracellular, no TM Sosui no TM, soluble protein TMHMM N terminus extracellular, no TM Signal Peptide Signal P none pI pI/MW tool 4.49 Molecular weight pI/MW tool 20.16 kDa Localization PSORT 64% peroxisome, 45% cyto plasmic, 15.3% lysosome PSORT II 52.2% cytoplasmic, 34.8% nuclear Motifs Pfam none Prints none Blocks none

TABLE XXIIIA Exon compositions of 158P3D2 var1 Exon Number Start End Exon 1 1 836 Exon 2 837 922 Exon 3 923 1021 Exon 4 1022 1263 Exon 5 1264 1547 Exon 6 1548 1648 Exon 7 1649 1961

TABLE XXIIIB Exon compositions of 158P3D2 var2 Exon Number Start End Exon 1 1 95 Exon 2 96 138 Exon 3 139 239 Exon 4 240 377 Exon 5 378 494 Exon 6 495 623 Exon 7 624 1835 Exon 8 1836 1921 Exon 9 1922 2020 Exon 10 2021 2222 Exon 11 2223 2506 Exon 12 2507 2607 Exon 13 2608 2918

TABLE XXIV Nucleotide sequence of transcript variant 158P3D2 var2 (Seq. ID No. 6266) atcaaggccc tgggctggag gaagacatcc cagatccaga ggagctcgac tgggggtcca 60 agtactatgc gtcgctgcag gagctccagg ggcagcacaa ctttgatgaa gatgaaatgg 120 atgatcctgg agattcagat ggggtcaacc tcatttctat ggttggggag atccaagacc 180 agggtgaggc tgaagtcaaa ggcactgtgt ccccaaaaaa agcagttgcc accctgaaga 240 tctacaacag gtccctggag gaagaattta accactttga agactggctg aatgtgtttc 300 ctctgtaccg agggcaaggg ggccaggatg gaggtggaga agaggaagga tctggacacc 360 ttgtgggcaa gttcaagggc tccttcctca tttaccctga atcagaggca gtgttgttct 420 ctgagcccca gatctctcgg gggatcccac agaaccggcc catcaagctc ctggtcagag 480 tgtatgttgt aaaggctacc aacctggctc ctgcagaccc caatggcaaa gcagaccctt 540 acgtggtggt gagcgctggc cgggagcggc aggacaccaa ggaacgctac atccccaagc 600 agctcaaccc catctttgga gagatcctgg agctaagcat ctctctccca gctgagacgg 660 agctgacggt cgccgtattt gaacatgacc tcgtgggttc tgacgacctc atcggggaga 720 cccacattga tctggaaaac cgattctata gccaccacag agcaaactgt gggctggcct 780 cccagtatga agtgtgggtc cagcagggcc cacaggagcc attctgagtt tctggccaaa 840 cacattcaag ctcacattcc cttttgtgtc tccagatcct atgatttcat ggaaggggac 900 cctcccaccc accgccactg ccaaccaaga catagctcag tggtcaagac ttgggcttgg 960 gagtcgggat cctgtaacga atgtcacttg accgctttct ttttttatga aacagtctcg 1020 ctctgtctcc caggttggag tgcagtggca cgatctcggc tgactgcaac ctccacctcc 1080 tgggttcaag cgattctcct gcctcagcct ccccagtagc tgggattaca ggcgtgggcc 1140 cccatgtcca gctaattttt atattttcgc tctgtctccc aggttggagt gcagtggcac 1200 gatctcggct gactgcaacc tccacctcct gggttcaagc gattctcctg cctcagcctc 1260 cccagtagct gggattacag gcgtgggccc ccatgtccag ctaattttta tatttttagt 1320 agagacaggg tttcaccatg ttgtccaggc tggtcttgaa cccctgacct caagtgatcc 1380 acccacctct gcctcccaaa gtgctgggat tacaggtgtg agccaccatg ccaggccctc 1440 ttaacctctt caagtctgtt ttctcatctg caaaacagag gtaataagat cagtatcttc 1500 ttaatggaag cacctgggct acattttttt cattcattgt tatcataaat gaggactaac 1560 ctgtctcccg ttgggagttt tgaacctaga cctcatgtct tcatgacgtc atcactgccc 1620 caggcccagc tgtgtcccta caccaqcccc agctgacgca tcttcttttt ctgcctgtag 1680 agatggttac aatgcctggc gtgatgcatt ctggccttcg cagatcctgg cggggctgtg 1740 ccaacgctgt ggcctccctg cccctgaata ccgagccggt gctgtcaagg tgggcagcaa 1800 agtcttcctg acaccaccgg agaccctgcc cccagggatc tcttcacatg tggattgaca 1860 tctttcctca agatgtgcct gctccacccc cagttgacat caagcctcgg cagccaatca 1920 gctatgagct cagagttgtc atctggaaca cggaggatgt ggttctggat gacgagaatc 1980 cactcaccgg agagatgtcg agtgacatct atgtgaagag ctgggtgaag gggttggagc 2040 atgacaagca ggagacagac gttcacttca actccctgac tggggagggg aacttcaatt 2100 ggcgctttgt gttccgcttt gactacctgc ccacggagcg ggaggtgagc gtctggcgca 2160 ggtctggacc ctttgccctg gaggaggcgg agttccggca gcctgcagtg ctggtcctgc 2220 aggatccctg gagttgcagc taccagacat ggtgcgtggg gcccggggcc ccgagctctg 2280 ctctgtgcag ctggcccgca atggggccgg gccgaggtgc aatctgtttc gctgccgccg 2340 cctgaggggc tggtggccgg tagtgaagct gaaggaggca gaggacgtgg agcgggaggc 2400 gcaggaggct caggctggca agaagaagcg aaagcagagg aggaggaagg gccggccaga 2460 agacctggag ttcacagaca tgggtggcaa tgtgtacatc ctcacgggca aggtggaggc 2520 agagtttgag ctgctgactg tggaggaggc cgagaaacgg ccagtgggga aggggcggaa 2580 gcagccagag cctctggaga aacccagccg ccccaaaact tccttcaact ggtttgtgaa 2640 cccgctgaag acctttgtct tcttcatctg gcgccggtac tggcgcaccc tggtgctgct 2700 gctactggtg ctgctcaccg tcttcctcct cctggtcttc tacaccatcc ctggccagat 2760 cagccaggtc atcttccgtc ccctccacaa gtgactctcg ctgaccttgg acactcaccc 2820 agggtgccaa cccttcaatg cctgctcctg gaagtctttc ttacccatgt gagctacccc 2880 agagtctagt gcttcctctg aataaaccta tcacagcc 2918

TABLE XXV Nucleotide sequence alignment of 158P3D2 var1 (Seq. ID No. 6267) and 158P3D2 var2 (Seq. ID No. 6268)

TABLE XXVI Peptide sequences of protein coded by 158P3D2 var2 >158P3D2 var2a (Seq. ID NO. 6269) MDDPGDSDGV NLISMVGEIQ DQGEAEVKGT VSPKKAVATL KIYNRSLEEE FNHFEDWLNV 60 FPLYRGQGGQ DGGGEEEGSG HLVGKFKGSF LIYPESEAVL FSEPQISRGI PQNRPIKLLV 120 RVYVVKATNL APADFNGKAD PYVVVSAGRE RQDTKERYIP KQLNPIFGEI LELSISLPAE 180 TELTVAVFEH DLVGSDDLIG ETHIDLENRF YSHHRANCGL ASQYEVWVQQ GPQEPF 236 >158P3D2 VAR2b (Seq. ID No. 6270) MVRGARGPEL CSVQLARNGA GPRCNLFRCR RLRGWWPVVK LKEAEDVERE AQEAQAGKKK 60 RKQRRRKGRP EDLEFTDMGG NVYILTGKVE AEFELLTVEE AEKRPVGKGR KQPEPLEKPS 120 RPKTSFNWFV NPLKTFVFFI WRRYWRTLVL LLLVLLTVFL LLVFYTIPGQ ISQVIFRPLH 180 K 181

TABLE XXVII Amino acid sequence alignment of 158P3D2 var1 (Seq. ID No. 6271) and 158P3D2 var2 (Seq. ID No. 6272) Score = 372 bits (956), Expect = e−103Identities = 181/181 (100%), Positives = 181/181 (100%) Query: 148 MVRGARGPELCSVQLARNGAGFRCNLFRCRRLRGWWPVVKLKEAEDVEREAQEAQAGKKK 207 MVRGARGPELCSVQLARNGAGPRCNLFRCRRLRGWWPVVKLKEAEDVEREAQEAQAGKKK Sbjct: 1 MVRGARGPELCSVQLARNGAGPRCNLFRCRRLRGWWPVVKLKEAEDVEREAQEAQAGKKK 60 Query: 208 RKQRRRKGRPEDLEFTDMGGNVYILTGKVEAEFELLTVEEAEKRPVGKGRKQPEPLEKPS 267 RKQRRRKGRPEDLEFTDMGGNVYILTGKVEAEFELLTVEEAEKRPVGKGRKQPEPLEKPS Sbjct: 61 RKQRRRKGRPEDLEFTDMGGNVYILTGKVEAEFELLTVEEAEKRPVGKGRKQPEPLEKPS 120 Query: 268 RPKTSFNWFVNPLKTFVFFIWRRYWRTLVLLLLVLLTVFLLLVFYTIPGQISQVIFRPLH 327 RPKTSFNWFVNPLKTFVFFIWRRYWRTLVLLLLVLLTVFLLLVFYTIPGQISQVIFRPLH Sbjct: 121 RPKTSFNWFVNPLKTFVFFIWRRYWRTLVLLLLVLLTVFLLLVFYTIPGQISQVIFRPLH 180 Query: 328 K 328 K Sbjct: 181 K 181 Note: Protein variant 158P3D2 var2a does not share common sequence with protein 158P3D2 var1. 

1. A method of inhibiting growth of cancer cells that express 158P3D2, comprising: administering to said cells an antibody or fragment thereof, either of which specifically bind to a 158P3D2 protein of SEQ ID NO:6275, thereby inhibiting the growth of said cells.
 2. The method of claim 1 wherein said antibody is a single chain monoclonal antibody, or fragment thereof.
 3. The method of claim 1, wherein said antibody or fragment is a recombinant protein comprising the antigen-binding region of an antibody that specifically binds to 158P3D2 protein.
 4. The method of claim 1, wherein said antibody or fragment is a human antibody or fragment.
 5. The method of claim 1, wherein said antibody or fragment is conjugated with a cytotoxic agent. 